impact of plasmin activity on the shelf life and...
TRANSCRIPT
Impact of plasmin activity on the shelf life
and stability of UHT milk
PhD thesis by
Valentin Maximilian Rauh
August 2014
Aarhus University
Faculty of Science and Technology
Department of Food Science
Blichers Allé 20
8830 Tjele
Denmark
Arla Strategic Innovation Center
Milk Science & Functionality
Protein Chemistry
Rørdrumvej 2
8220 Brabrand
Denmark
I
Preface
This PhD thesis concludes three years’ work at Arla Strategic Innovation Center and the
Department of Food Science, Aarhus University. This industrial PhD project was a collaboration
between Aarhus University, Arla Foods, Copenhagen University and Lund University. As part of
the PhD study, a three months stay at Copenhagen University, Department of Food Science was
carried out.
Main supervisor
Associate Professor Marianne Hammershøj
Department of Food Science, Aarhus University
Co-supervisors
Professor Lotte B. Larsen
Department of Food Science, Aarhus University
Professor Richard Ipsen
Department of Food Science, Copenhagen University
Professor Marie Paulsson
Department of Food Technology, Lund University
Mette Bakman
Arla Strategic Innovation Center, Arla Foods
Assessment committee
Senior researcher Niels Oksbjerg (chairman)
Department of Food Science, Aarhus University
Dr. Thom Huppertz
Dairy and Ingredient Technology, NIZO food research B.V., Ede, The Netherlands
Prof. Dr.-Ing. Habil. Jörg Hinrichs
Institute of Food Science and Biotechnology, Universität Hohenheim, Stuttgart, Germany
II
Acknowledgements
This industrial PhD project was made possible by funding from the Danish Agency for Science,
Technology and Innovation (DASTI) and Arla Foods.
I would like to thank my university supervisors Marianne Hammershøj, Lotte B. Larsen, Richard
Ipsen and Marie Paulsson for the valuable discussions and support during the project. Each one of
you contributed with a different specialization and experience, so that every aspect of the project,
from processing, enzymes and dairy chemistry to statistics and paper writing, was covered.
The innovation department of Arla Foods underwent major changes in the last three years. With
these changes, new people came into the project and others had to leave. I would like to thank all of
my Arla supervisors, Carola Edsman, Anja Sundgren, Mette Bakman, Jacob H. Nielsen and Grith
Mortensen for the support during the project. A special thanks to Mette, Anja and Grith for being
great managers and always believing in me and the project.
I would like to thank the protein chemistry team in the Arla Strategic Innovation Center, Vibeke
Mortensen, Betina Hansen, Lene B. Johansen and Mette Christensen for the countless hours of
listening to and discussing results, ideas, hypotheses and the assistance with the analysis. A special
thanks to Lene for introducing and teaching me the wonderful world of mass spectrometry; without
this the project would have looked very different! I also would like to thank all colleagues in the
Arla Strategic Innovation Center, both in Stockholm and Brabrand, for teaching me new techniques,
helping me with analysis and conducting the pilot plant trials.
Thanks to my fellow PhD students both at Aarhus University, Copenhagen University and Arla for
fruitful discussions and a great working environment. Thilo Berg is thanked for proofreading the
thesis. Thanks to Nanna S. Villumsen and Therese Jansson for great scientific discussions and
motivational talks.
I would like to thank my family and friends in Germany, Denmark and Sweden for a great time, for
motivating me and showing me that there is a life outside the PhD. Thank you Vera and Thilo for
your hospitality and always making me feel at home when I was in Stockholm and Copenhagen.
Valentin Rauh, August 2014
III
Abstract
Milk production in Europe is increasing, while the markets for dairy products are saturated and milk
consumption is decreasing. The production of high quality long shelf life products will enable the
dairy industry to distribute products into new markets and increase export into existing growth
markets. Ultra high temperature (UHT) treatment of milk aims to give a commercially sterile
product with a shelf life of three months or more without refrigeration. The shelf life of UHT milk
is determined by deterioration of flavour and functional properties, which can be caused by either
heat induced chemical changes or the activity of enzymes in milk. The principal enzyme in good
quality milk which can survive UHT treatments is the indigenous protease plasmin. Plasmin is the
active part of an enzyme system consisting of the inactive precursor plasminogen and several
activators and inhibitors, which can interact with different milk constituents.
The main objective of this PhD project was to investigate the effect of residual plasmin activity on
physical-chemical changes in UHT milk.
Due to the complexity of the plasmin and milk system, interpretation of plasmin activity
measurement results can be difficult. In the first part of this project, a spectrophotometric assay for
plasmin and plasminogen derived activity was optimized and extended to turbid samples and
allowed measurement of plasmin activity in fat containing milk samples. The measured plasmin
activity was largely affected by the sample preparation and assay conditions. The developed assay
reduces the risk of underestimation of plasmin activity in milk and can be modified for different
samples and purposes.
The effect of residual plasmin activity on physical-chemical changes in milk during storage at 20 ºC
were studied in direct steam infusion heat treated milk with ultra-short holding times. Plasmin
activity resulted in extensive proteolysis of caseins and a bitter off flavour appeared after six weeks
of storage at which time more than 60 % of αS- and β-caseins were hydrolysed. After 10 weeks of
storage more than 90 % of αS- and β-caseins were hydrolysed and the milk began to form a gel.
Bitterness was found to be the most important shelf life limiting factor for milk with residual
plasmin activity.
Identification of peptides in this UHT milk with residual plasmin activity allowed the exploration of
the proteolytic pathway of plasmin in a milk system. 66 casein derived peptides were formed by the
plasmin, of which 23 could have been responsible for the observed bitterness. Plasmin showed the
IV
highest affinity for the hydrophilic regions in the caseins and the least affinity for hydrophobic and
phosphorylated regions.
Protein lactosylation, the initial stage of the Maillard reaction, was studied in different UHT milks
over a storage period of six months by liquid chromatography-mass spectrometry (LC-MS) and the
common marker furosine. The initial furosine concentration in direct steam infusion heat treated
milk was found to be 10-15 times lower as compared to an indirectly processed UHT milk, but no
significant difference in the increase of furosine concentration during storage was found between
the milks. A quantitative correlation lactosylation measured by LC-MS and furosine concentration
was established, allowing for a simpler and faster analysis of protein lactosylation.
The results presented in this thesis expand our understanding of the plasmin system in milk and can
help to control plasmin activity in UHT milk. This gives a direct way to improve the quality and
shelf life of UHT milk products. Furthermore, the fundamental findings of this thesis can be
transferred and applied to other dairy products.
V
Sammendrag (abstract in Danish)
Mælkeproduktion i Europa er stigende, samtidig er markedet for mejeriprodukter mættet og
forbruget af mælk er faldende. Fremstilling af højkvalitetsprodukter med lang holdbarhed vil gøre
det muligt for mejeriindustrien at lancerede mejeriprodukter på nye markeder og samtidig øge
eksporten til eksisterende vækstmarkeder.
Formålet med ultra høj temperatur (UHT) behandling af mælk er at opnå et kommercielt sterilt
produkt med en holdbarhed på minimum tre måneder udenfor køl. Holdbarheden af UHT mælk
begrænses af smagsforringelse og ændrede funktionelle egenskaber, der kan være forårsaget af
enten varmeinducerede kemiske ændringer eller restaktivitet af mælkens naturlige enzymer. Det
vigtigste enzym i mælk af god kvalitet er den naturligt forekommende protease plasmin, der kan
overleve UHT behandlingen. Plasmin er den aktive del af et enzymsystem, der består af den
inaktive proenzym, plasminogen, samt forskellige aktivatorer og inhibitorer, der kan interagere med
de forskellige komponenter i mælken.
Hovedformålet med dette PhD projekt var at undersøge effekten af restplasminaktivitet på fysisk-
kemiske ændringer af UHT mælk.
Tolkning af plasminaktivitetsmålinger kan være vanskelig på grund af kompleksiteten af plasmin-
og mælkesystemet. I den første del af dette projekt blev en spektrofotometrisk metode til måling af
plasmin og plasminogenafledt aktivitet optimeret og udvidet til at kunne måle turbide
prøvematricer, hvilket muliggør måling af plasminaktivitet i fedtholdige mælkeprøver. Den målte
plasminaktivitet blev i høj grad påvirket af prøveforberedelsesmetoden og målevilkår.
Udviklimngen af metoden reducerede risikoen for underestimering af plasminaktivitet i mælk og
kan modificeres til måling af forskellige prøver under forskellige vilkår.
Effekten af restplasminaktivitet på fysisk-kemiske ændringer i mælk opbevaret ved 20 °C blev
undersøgt. Mælken blev varmebehandlet med direkte dampinfusion i ultrakort tid. Aktiviteten af
plasmin resulterede i omfattende proteolysis af kaseiner og dannelse af bitter afsmag efter seks
ugers opbevaring, hvor mere end 60% af S- og β-kasein var hydrolyseret. Efter 10 ugers
opbevaring var mere end 90% af S- og β-kasein hydrolyseret og mælken begyndte at gelere. Bitter
smag blev fundet at være den vigtigste holdbarhedsbegrænsende faktor for mælk indeholdende
restplasminaktivitet.
Identifikation af peptider i UHT mælk med restplasminaktivitet gjorde det muligt, at udforske
VI
plamins proteolytiske nedbrydningsmønster i mælk. 66 kaseinafledte peptider blev dannet af
plasmin, hvoraf 23 kan være årsag til den observerede bitterhed. Plasmins affinitet overfor
spaltningssteder i kasein er anderledes i UHT mælk sammenlignet med kaseinmodelsystemer.
Plasmin viste højest affinitet overfor de hydrofile regioner i kasein og mindst affinitet overfor de
hydrofobe og fosforylerede regioner.
Laktosylering af proteiner, der er den indledende fase af Maillard-reaktionen, blev undersøgt i
forskellige UHT mælkeprøver over en lagrignsperiode på seks måneder ved anvendelse af
væskekromatografi koblet med massespektrometri (LC-MS) og markøren furosin. Den indledende
furosinkoncentration i mælk varmebehandlet med direkte dampinfusion blev målt til at være 10-15
gange lavere end indirekte UHT behandlet mælk, men der blev ikke påvist en signifikant forskel i
stigningen af furosin under lagring. En kvantitativ korrelation mellem laktosylering målt ved LC-
MS og furosinkoncentrationen blev etableret.
Resultaterne præsenteret i denne afhandling har udvidet forståelsen af plasminsystemet i mælk og
kan bidrage til at kontrollere plasminaktivitet i UHT mælk. På denne måde kan der opnås en
forbedret kvalitet samt holdbarhed af UHT produkter. Yderlig kan de fundamentale resultater fra
denne afhandling overføres og anvendes på andre mejeriprodukter.
VII
Zusammenfassung (abstract in German)
Die Milchproduktion in Europa nimmt stetig zu, während der Markt für Molkereiprodukte gesättigt
und der Milchkonsum rückläufig ist. Die Produktion von qualitativ hochwertigen Produkten mit
langer Haltbarkeit ermöglicht der Milchindustrie neue Märkte zu erschließen und den Export in
bereits existierende Wachstumsmärkte zu erhöhen. Das Ziel der Ultrahocherhitzung (UHT) von
Milch ist ein kommerziell steriles Produkt mit einer Haltbarkeit von drei Monaten oder länger. Die
Haltbarkeit von UHT Milch wird durch eine Verschlechterung des Geschmacks oder funktioneller
Eigenschaften beinflusst, die entweder durch Hitze bedingte chemische Veränderungen oder
Aktivität von Enyzmen in der Milch verursacht werden kann. Die endogene Protease Plasmin ist
das wichtigste Enyzm in Milch von hoher Qualität, das die UHT Behandlung überstehen kann.
Plasmin ist der aktive Teil eines Enzymsystems, das aus dem inaktivem Vorläufer Plasminogen
sowie einigen Aktivatoren und Inhibitoren besteht, die mit verschiedenen Milchkomponenten
interagieren können.
Das Hauptziel dieses Projektes war die Untersuchung des Einflusses der Plasminaktivität auf
physikalische und chemische Veräderungen in UHT Milch.
Die Interpretation von Plasminaktivitätmessungen gestaltet sich aufgrund der Komplexität der
Milch und des Plasminsystems als schwierig. Der erste Teil dieses Projektes beschäftigte sich daher
mit der Optimierung einer spektrophotometrischen Methode zur Messung von Plasmin- und
Plasminogenaktivität. Die Methode wurde außerdem erweitert, sodass Messungen von trüben
Proben möglich waren, was die Analyse von fetthaltigen Milchproben erlaubte. Die gemessene
Plasminaktivität hing stark von der Probenaufbereitung und Analysekonditionen ab. Die
entwickelte Methode reduziert das Risiko Plasminaktivität in Milch zu unterschätzen und kann
darüberhinaus für verschiedene Proben und Zwecke modifiziert warden.
Der Effekt von Plasminaktivität auf physikalische und chemische Veränderungen in UHT Milch
wärend der Lagerung bei 20 ºC wurde in einer Milch untersucht, die mit direkter Dampfinfusion mit
extrem kurzer Haltezeit erhitzt wurde. Plasminaktivität führte zu erheblicher Proteolyse von
Caseinen und ein bitterer Geschmack trat nach sechs Wochen Lagerung auf. Zu diesem Zeitpunkt
waren mehr als 60 % der αS- and β-Caseine hydrolysiert. Nach 10 Wochen Lagerung waren mehr
als 90 % der αS- and β-Caseine hydrolysiert und die Milch bildete ein Gel. Die Haltbarkeit der UHT
Milch wurde hauptsächlich durch den bitteren Geschmack limitiert.
VIII
Die Identifikation von Peptiden in der UHT Milch erlaubte eine Untersuchung des Proteolyseweges
von Plasmin in einem Milchsystem. 66 von Caseinen abstammende Peptide wurden von Plasmin
gebildet und 23 von diesen könnten den beobachteten bitteren Geschmack verursacht haben. Eine
abweichende Affinität von Plasmin für Spaltstellen in der UHT Milch und Casein Modelsystemen
wurde festgestellt. Plasmin zeigte die höchste Affinität für hydrophile Regionen der Caseine und die
geringste Affinität für hydrophobe und phosphorylierte Regionen.
Die erste Phase der Maillard Reaktion, die Laktosylierung von Proteinen, wurde in verschiendenen
UHT Milchtypen über einen Zeitraum von sechs Monaten mit Hilfe von Flüssigchromatographie
mit Massenspektrometrie-Kopplung (LC-MS) sowie der Analyse der Furosinkonzentration
untersucht. Die anfängliche Furosinkonzentration in Milch, die durch direkte Dampfinfusion erhitzt
wurde, war 10-15 mal niedriger im Vergleich zu indirekt erhitzer UHT Milch. Es wurden jedoch
keine signifikanten Unterschiede in der Zunahme der Furosinkonzentration während der Lagerung
in den UHT Milchtypen gefunden. Eine quantitative Korrelation zwischen der mit LC-MS
gemessenen Laktosylierung und der Furosinkonzentration konnte hergestellt werden, was die
Analyse der Proteinlaktosylierung vereinfacht und verkürzt.
Die Ergebnisse in der vorliegenden Arbeit erweitern das Verständins des Plasminsystems in Milch
und können dazu beitragen, Plasminaktivität in UHT Milch zu kontrollieren. Dadurch kann sowohl
die Qualtität als auch die Haltbarkeit von UHT Milchprodukten verbessert werden. Darüber hinaus
können die grundlegenden Erkenntnisse dieser Arbeit auf andere Milchprodukte übertragen und
angewendet werden.
IX
Table of contents
Preface ................................................................................................................................................... I
Acknowledgements ............................................................................................................................. II
Abstract .............................................................................................................................................. III
Sammendrag (abstract in Danish) ....................................................................................................... V
Zusammenfassung (abstract in German).......................................................................................... VII
List of included publications ............................................................................................................ XII
List of abbreviations........................................................................................................................ XIII
1 Introduction .................................................................................................................................. 1
2 Project overview .......................................................................................................................... 2
2.1 Aim and objectives ................................................................................................................ 2
2.2 Experimental outline ............................................................................................................. 2
2.3 Overview of methods ............................................................................................................ 3
3 Summary of included publications .............................................................................................. 6
3.1 Paper I: The determination of plasmin and plasminogen-derived activity in turbid samples
from various dairy products using an optimised spectrophotometric method ................................. 6
3.2 Paper II: Plasmin activity as a possible cause for age gelation in UHT milk produced by
direct steam infusion ........................................................................................................................ 6
3.3 Paper III: Plasmin Activity in UHT Milk: Relationship between Proteolysis, Age Gelation,
and Bitterness ................................................................................................................................... 7
3.4 Paper IV: Protein lactosylation in UHT milk during storage measured by liquid
chromatography-mass spectrometry and furosine ........................................................................... 7
4 The plasmin system in milk ......................................................................................................... 8
4.1 Indigenous enzymes in milk .................................................................................................. 8
4.2 The plasmin system in milk .................................................................................................. 8
4.2.1 Components and localisation of the plasmin system in milk ......................................... 8
4.2.2 Molecular features of plasmin and plasminogen ......................................................... 10
X
4.2.3 Activation of plasminogen ........................................................................................... 12
4.2.4 Inhibitors of plasmin and plasminogen activators ....................................................... 13
4.2.1 Specificity of plasmin .................................................................................................. 14
4.2.2 Factors affecting the plasmin system ........................................................................... 18
4.2.2.1 Environmental factors ........................................................................................... 18
4.2.2.2 Effect of milk processing on the plasmin system ................................................. 19
5 Measurement of the plasmin system activity ............................................................................. 23
5.1 Activity assays ..................................................................................................................... 23
5.1.1 Plasmin and plasminogen............................................................................................. 23
5.1.1.1 Effect of sample preparation on plasmin activity ................................................. 24
5.1.1.2 Effect of sample composition and buffers on plasmin activity ............................ 25
5.1.1.3 Comparison of activity assays .............................................................................. 27
5.1.2 Plasminogen activators ................................................................................................ 30
5.2 Immunoassays ..................................................................................................................... 30
5.3 Other assays ......................................................................................................................... 31
5.4 Proteolysis ........................................................................................................................... 32
5.4.1 Hydrolysis of caseins ................................................................................................... 32
5.4.2 Peptide formation ......................................................................................................... 33
5.4.3 Kinetic aspects of proteolysis during storage .............................................................. 34
6 UHT milk ................................................................................................................................... 39
6.1 Heat treatment of milk ......................................................................................................... 39
6.2 UHT milk processing .......................................................................................................... 39
6.2.1 Design of UHT processes ............................................................................................ 39
6.2.2 UHT processing systems .............................................................................................. 41
6.3 Heat-induced changes in milk ............................................................................................. 43
6.3.1 Proteins......................................................................................................................... 43
6.3.1.1 Whey proteins ....................................................................................................... 43
XI
6.3.1.2 Caseins .................................................................................................................. 45
6.3.2 Heat inactivation of the plasmin system ...................................................................... 45
6.3.3 Effect of cysteine on plasmin and milk proteins during heat treatment ....................... 49
6.3.4 Maillard reaction .......................................................................................................... 52
6.3.5 Lactulose ...................................................................................................................... 53
6.4 Shelf life development of UHT milk ................................................................................... 54
6.4.1 Age gelation of UHT milk ........................................................................................... 54
6.4.2 Flavour development.................................................................................................... 61
6.4.2.1 Chemical changes affecting flavour development ................................................ 61
6.4.2.2 Effect of enzymes on flavour ................................................................................ 62
6.4.3 Lactosylation of milk proteins during storage of UHT milk ....................................... 64
7 Conclusions ................................................................................................................................ 68
8 Perspectives ................................................................................................................................ 70
9 References .................................................................................................................................. 72
XII
List of included publications
This thesis is mainly based on the papers listed below and are referred to in the text by their roman
numerals. The papers are included in the back of the thesis.
Paper I
Rauh, V. M., Bakman, M., Ipsen, R., Paulsson, M., Kelly, A. L., Larsen, L. B., & Hammershøj, M.
(2014). The determination of plasmin and plasminogen-derived activity in turbid samples from
various dairy products using an optimised spectrophotometric method. International Dairy Journal,
38, 74-80.
Paper II
Rauh, V. M., Sundgren, A., Bakman, M., Ipsen, R., Paulsson, M., Larsen, L. B., & Hammershøj, M.
(2014). Plasmin activity as a possible cause for age gelation in UHT milk produced by direct steam
infusion. International Dairy Journal, 38, 199-207.
Paper III
Rauh, V. M., Johansen, L. B., Ipsen, R., Paulsson, M., Larsen, L. B., & Hammershøj, M. (2014).
Plasmin Activity in UHT Milk: Relationship between Proteolysis, Age Gelation, and Bitterness.
Journal of Agricultural and Food Chemistry, 62, 6852-6860.
Paper IV
Rauh, V. M., Johansen, L. B., Ipsen, R., Paulsson, M., Larsen, L. B., & Hammershøj, M. (2014).
Protein lactosylation in UHT milk during storage measured by liquid chromatography-mass
spectrometry and furosine. Manuscript intended for International Journal of Dairy Technology.
XIII
List of abbreviations
AU arbitrary unit
α-la α-lactalbumin
β-lg β-lactoglobulin
BSA bovine serum albumin
cn casein
CLSM confocal laser scanning microscopy
EDTA ethylenediaminetetraacetic acid
ELISA enzyme-linked immunosorbent assay
EACA ε-amino caproic acid
ESI-MS electrospray ionisation mass spectrometry
FAST fluorescence of advanced Maillard products and soluble tryptophan
HMF 5-hydroxylmethyl-2-furfural
(RP-) HPLC (reverse phase) high performance liquid chromatography
LC liquid chromatography
MS mass spectrometry
NCN non-casein nitrogen
MALDI MS matrix assisted laser desorption mass spectrometry
MFGM milk fat globule membrane
MS mass spectrometry
PA plasminogen activator
PL plasmin
PP proteose peptone
TCA trichloroacetic acid
tPA tissue-type plasminogen activator
UHT ultra high temperature
uPA urokinase-type plasminogen activator
UV ultra violet
1
1 Introduction
Almost all consumption milk is subjected to heat treatment during processing to inactivate
pathogenic and spoilage microorganisms and to prolong the shelf life of the milk. Heat treatment
of milk is always a balance between the inactivation of undesired components, i.e.
microorganisms and enzymes, the preservation of desired properties and negative heat induced
chemical effects. Depending on the severity of the heat treatment, the shelf life of milk is
therefore limited by a decrease in organoleptic and functional properties, which can be caused by
either spoilage microorganisms and enzymes or heat induced changes in milk. Low pasteurized
milk (72 °C for 15-30 s) has an excellent flavour, however, the shelf life is only one to two weeks
when refrigerated due to deterioration by spoilage microorganisms. On the other hand, UHT milk
(135-150 °C for 1-10 s) is commercially sterile and has a shelf life from two months up to one
year without refrigeration, but the flavour of the milk is significantly changed and further
deteriorates during storage resulting in a low acceptance for UHT milk by consumers.
The quality of UHT milk can be improved by optimisation of the heat treatment process. Direct
UHT processes use steam to rapidly heat up the milk to the desired temperature. Thereafter the
milk is rapidly cooled in a vacuum chamber. In indirect UHT processes, milk is heated in a
tubular or plate heat exchanger by hot water and/or steam, which is separated from the milk by a
metal surface. Milk from direct steam UHT processes has an improved flavour compared to milk
from indirect UHT processes. The fast heating and cooling rates in direct steam processes reduce
the severity of the heat treatment in regards to chemical changes (Datta, et al., 2002).
Furthermore, the inactivation of microorganisms in the temperature range between 110-150 °C is
mainly dependent on the temperature, whereas chemical changes are mainly dependent on the
holding time when applying direct steam systems. Consequently, the heat treatment can be
optimized by increasing the temperature while decreasing the heating time. This leads to the
development of UHT processes outside the classic time temperature range operating at
temperatures > 150 °C combined with ultra-short holding times of < 0.2 s (Holst, et al., 2010;
Holst, et al., 2012; Huijs, et al., 2004). These processes enable the production of commercially
sterile products with minimum chemical changes, but are not able to sufficiently inactivate heat
resistant enzymes to ensure a long shelf life.
Enzymes in milk are either indigenous enzymes already present in milk or are produced by
bacteria in milk before processing. Of these enzymes, heat resistant proteolytic and lipolytic
enzymes from psychotrophic bacteria and the heat resistant major indigenous protease plasmin
have the largest impact on product deterioration during storage of this type of UHT milk, causing
2
off flavours and gelation (Datta & Deeth, 2003; McKellar, et al., 1984). While the problem of
bacterial enzymes can largely be avoided by using high quality raw milk and minimizing the
growth of bacteria in milk before processing, there is no possibility to eliminate plasmin in milk.
An additional pre or post-heat treatment is usually applied to inactivate plasmin, which on the
other hand will decrease the organoleptic quality of the milk (van Asselt, et al., 2008).
While the heat inactivation of plasmin has been studied intensively, the effect of plasmin activity
and proteolysis on UHT milk quality and the underlying mechanisms are only known to a limited
extent. The main hypothesis of this project is that the shelf life of UHT milk with residual
plasmin activity can be controlled, when proteolysis and quality defects by plasmin can be
correlated. The second hypothesis is that the shelf life of the UHT milk can be predicted and
optimised by correlating plasmin activity measured in an assay with the occurring proteolysis.
2 Project overview
2.1 Aim and objectives
The main objective of the project is to investigate plasmin mediated proteolysis and quality
defects during the storage of UHT milk. The second objective is to correlate the proteolysis with
plasmin activity measurements. Since plasmin is the active part of a complex enzyme system in a
complex milk system, the measurement of plasmin activity depends largely on the used assay.
The first objective is therefore to investigate the factors influencing plasmin activity in an assay
and to develop a new or to improve existing assays.
2.2 Experimental outline
It was initially planned to study plasmin activity in whey and micellar casein besides milk. For
this, a diafiltration experiment was conducted to obtain whey and casein fractions and to
investigate plasmin activity during diafiltration. Due to lack of time and considerable process
variation, it was decided to not further pursue this and the focus was put on milk only. The whey
and casein fractions were used for initial testing of the plasmin and plasminogen derived activity
assay. Different factors influencing the plasmin activity in different sample matrices were
investigated during the method development of the plasmin activity assay.
A pilot scale UHT experiment was carried out at the Arla Foods pilot plant in Stockholm. In total,
five UHT milks, four processed with direct steam infusion and ultra-short holding times and
varying pre-heat treatment and one indirectly heat treated UHT milk as reference were produced.
The trial was performed in triplicate on three consecutive weeks. The milk was transported to the
3
Arla Foods Strategic Innovation Center in Denmark and the milk was stored at 20 °C and
analysed on a weekly basis for 14-16 weeks. It was aimed for that the 4 directly processed UHT
milk samples should contain four different levels of plasmin activity. However, the UHT
treatment resulted in two UHT milks with similar plasmin activity and two UHT milks without
plasmin activity. A correlation of proteolysis and plasmin activity was therefore not possible. The
UHT milks without plasmin activity were further analysed on a monthly basis up to a storage
time of six months to investigate chemical changes and protein modification during storage.
2.3 Overview of methods
A summary of the methods used in this project is given in Table 1. The detailed descriptions of
the methods can be found in the included publications. The paper in which the method is
described is annotated in the table.
Table 1: Overview of methods used in the project with the aim of the analysis and a short description of the principle. The paper in which the methods are described in detail are indicated in the table.
Analysed property Aim Method Principle Paper
Plasmin activity Determination of plasmin
activity
Activity assay Plasmin activity is measured by the release of p-nitroaniline upon
hydrolysis of the substrate S-2251 over time using a spectrophotometer.
I & II
Plasminogen derived
activity
Determination of
plasminogen derived
activity
Activity assay Plasminogen derived activity is measured after activation of
plasminogen by uPA in the same way as plasmin activity.
I & II
Colour Measurement of colour
changes in milk during
storage
Colorimeter Colour was measured using the Lab colour space. It consists of a
lightness value L (scale ranges from 0 to 100, where higher L values
equals whiter colour), the a value for the red-green axis (scale ranges
from -100 to 100, where positive a value equals red and negative a
values equals green) and the b value for the yellow-blue axis (scale
ranges from -100 to 100, where positive b value equals yellow and
negative b value equals blue).
II
Particle size Investigation of particle size
changes
Static light
scattering
Static light scattering uses the diffraction pattern of light from single
particles into different directions by multiple detectors in a dilution.
Particle size can be deduced from the diffraction pattern and the
refractive index
II
Microscopy Characterisation of gel
structure
Confocal laser
scanning
microscopy
(CLSM)
Gel particles are stained with the fluorophores FITC for protein and nile
red for fat which fluoresce green and red, respectively. The confocal
microscope allows high resolution images of the gel microstructure at
different depths of the sample and differentiation of fat and protein.
II
Viscosity Determination of viscosity
in relation to gelation
Rheometer Viscosity of milk was measured in a double cup system at 25 °C with a
constant shear rate of 100 s-1
for 120 s after a 15 s pre-shearing step.
II
Furosine Analysis of initial Maillard
reaction and protein
lactosylation
HPLC The protein attached Amadori product lactulosyllysine is converted to
furosine by acid hydrolysis at 115 °C and analysed by HPLC and diode
array detection.
IV
Table 1: (Continued)
Analysed property Aim Method Principle Paper
Protein analysis Measurement of protein
composition
LC-MS Milk proteins are dissociated by urea and sodium citrate, and reduced
by dithioerythritol. The proteins are separated by HPLC and detected by
MS and UV.
II
Analysis of whey protein
denaturation
LC-MS Caseins are precipitated by acidification to pH 4.6, and the soluble whey
proteins are separated by HPLC and detected by UV and MS.
Denaturation of whey proteins is measured by the difference in
concentration of pH 4.6 soluble whey proteins before and after heat
treatment.
II
Peptide analysis Identification of peptides LC-MS and
tandem MS
The pH 4.6 soluble peptides are separated by HPLC and analysed by
tandem MS. Both fixed fragmentation energies and dynamic
fragmentation energies based on charge state and mass were used for
peptide identification.
III
Identification of
polypeptides
LC-MS Polypeptides are identified by mass in the pH 4.6 soluble peptide
fraction and protein composition after deconvolution of the mass
spectra. Besides mass, phosphorylation patterns and genetic variants are
used for identification.
II
Protein modification Analysis of protein
lactosylation
LC-MS Proteins are dissociated, reduced and separated by RP-HPLC.
Lactosylation is either detected by deconvolution of the summed mass
spectra of a protein by a mass shift of +324 Da or directly in the
summed mass spectra as a mass shift of +324 Da divided by the charge
state of the investigated ion.
IV
Analysis of thiol-disulphide
rearrangements by cysteine
LC-MS Proteins are dissociated by urea and sodium citrate, but not reduced.
Rearrangement or thiol-disulphide interchange is characterized by the
appearance of monomeric protein and/or appearance of cysteine
attachment to proteins by a mass shift of +121 or +242 Da.
-
6
3 Summary of included publications
3.1 Paper I: The determination of plasmin and plasminogen-derived activity in
turbid samples from various dairy products using an optimised
spectrophotometric method
The objective of this study was to optimize and extend a spectrophotometric assay for plasmin
and plasminogen-derived activity to enable measurement of turbid samples and different dairy
products. The method was validated by assessing reproducibility, repeatability, level of detection
and recovery of plasmin activity in different sample matrices. The level of detection and
repeatability of this method were improved compared with previous spectrophotometric assays.
The second objective was to explore the effect of sample preparation and dissociation of plasmin
from caseins on plasmin activity. Skimming of the sample resulted in a decrease of plasmin
activity by 30% in pasteurised and homogenised whole milk, while skimming did not affect
plasmin activity in raw milk. Removal of the cream phase in homogenised milk samples by
skimming lead to an underestimation of plasmin activity. Comparison of pasteurised milk with a
micellar casein solution showed that the dissociation of plasmin and caseins by adding ε-amino
caproic acid (EACA) and NaCl decreased interference by caseins in the assay, but increases
inhibition of plasmin with serum-based inhibitory components.
3.2 Paper II: Plasmin activity as a possible cause for age gelation in UHT milk
produced by direct steam infusion
In this study, the effect of plasmin activity in direct steam infusion heat treated milk with ultra-
short holding times (>150 °C for <0.2 s) on physical-chemical changes in milk during storage
was investigated. Preheating at either 72 or 95 °C for 180 s was performed. Milk pre-heated at
72°C showed extensive proteolysis, but no proteolysis of κ-casein was detected. Plasmin was
identified as active protease and activation of plasminogen was observed as an increase in the rate
of casein hydrolysis during storage. A bitter off flavour appeared after six weeks of storage and
the milk contained less than 40% intact αS- and β-caseins when bitterness was noticed.
Proteolysis in the stored samples correlated with a decrease in pH and with changes in colour.
Gelation occurred after 10 weeks along with an increase in viscosity and almost complete
proteolysis of αS- and β-caseins. In conclusion, plasmin mediated proteolysis was involved in the
occurrence of age gelation and bitter off flavour. Bitterness preceded age gelation and was the
shelf-life limiting factor.
7
3.3 Paper III: Plasmin Activity in UHT Milk: Relationship between Proteolysis,
Age Gelation, and Bitterness
A peptidomic study on the directly heated UHT milk (>150 °C for <0.2 s) preheated at 72 °C for
180 s was performed to gain a deeper insight in the specificity and proteolytic pathway of
plasmin in a milk system in relation to age gelation and the formation of bitter peptides. Sixty-six
peptides from αS- and β-caseins could be attributed to plasmin activity during the storage period,
of which 23 were potentially bitter. Plasmin exhibited the highest affinity for the hydrophilic
regions in the caseins that most probably were exposed to the serum phase and the least affinity
for hydrophobic or phosphorylated regions. The proteolytic pattern observed suggests that
plasmin destabilizes the casein micelle by hydrolysing casein−casein and casein−calcium
phosphate interaction sites, which may subsequently cause age gelation in UHT milk.
3.4 Paper IV: Protein lactosylation in UHT milk during storage measured by
liquid chromatography-mass spectrometry and furosine
The initial stage of the Maillard reaction, protein lactosylation, occurs during the heat treatment
of milk and continues during subsequent storage. We compared the initial lactosylation and rate
of lactosylation during storage of milk proteins in directly and indirectly heated UHT milk using
liquid chromatography (LC) coupled with electron spray injection mass spectrometry (ESI-MS).
Furosine was used as an overall marker to allow a quantitative correlation of lactosylation
measured by LC-ESI-MS in the UHT milks. Protein lactosylation increased during the storage
period of 6 months at 20°C. The extent of protein lactosylation in the directly heated UHT milk
was approximately 10-15 times lower compared to indirectly heated UHT milk. The rate of
protein lactosylation during storage however was not significantly different for indirect and
directly heated UHT milk. Both the initial extent and the rate of lactosylation positively
correlated with the amount of lysine residues in the different proteins. A linear or exponential
correlation with furosine concentration could be established for minor and major lactosylated
proteins, respectively.
8
4 The plasmin system in milk
4.1 Indigenous enzymes in milk
Indigenous enzymes in milk have been subject to research for over a century and by now, about
70 enzymes have been identified in bovine milk (O’Mahony, et al., 2013). Indigenous enzymes,
as opposed to exogenous enzymes, originate from the cow itself. The indigenous enzymes in milk
originate from different sources. One source of enzymes is the blood plasma, and enzymes can
enter the milk by leaky junctions between mammary cells. Another source is the mammary
epithelial secretory cell cytoplasm. The milk fat globule membrane contains a large number of
enzymes as it originates from the apical membrane of the mammary cell, which itself originates
from the Golgi membranes. The last source of indigenous enzymes are somatic cells, which enter
the milk from blood during bacterial infections (O’Mahony, et al., 2013). While most of the
indigenous enzymes in milk do not have an obvious physiological role in milk, many of them can
have a significant effect on the quality and properties of dairy products (Kelly & Fox, 2006).
The enzymes most responsible for the deterioration of dairy products and relevant for the dairy
industry are lipases and proteases. Milk contains two protease systems, plasmin and cathepsin, of
which the first one is the principal protease in good quality milk, it has been extensively studied
due to its high heat stability and impact on dairy products (Kelly, et al., 2006). In the following
chapter, an overview of the plasmin system, factors affecting its action and its impact on dairy
products are given.
4.2 The plasmin system in milk
4.2.1 Components and localisation of the plasmin system in milk
The major indigenous milk protease plasmin (EC 3.4.21.7) is a serine protease with a pH
optimum of 7.5 at 37 °C (Grufferty & Fox, 1988b). Plasmin in milk is mainly present in its
inactive form, plasminogen, which can be activated by tissue-type (tPA) or urokinase type (uPA)
plasminogen activator (Bastian & Brown, 1996). The plasminogen concentration in fresh milk is
0.18-2.8 mg L-1
and 2-30 times higher compared to plasmin (0.1-0.7 mg L-1
) (Benfeldt, Connie,
et al., 1995; Ozen, et al., 2003; Richardson, B. C. & Pearce, 1981). Furthermore, the system is
regulated by the presence of inhibitors for plasmin and plasminogen activators (Figure 1).
Plasminogen is not expressed in the mammary gland and comparison of the amino acid sequence
of plasmin and plasminogen from blood and milk show these to be identical from the two sources
and it is thus demonstrated that plasmin originates from the blood (Benfeldt, Connie, et al., 1995;
Berglund, et al., 1995).
9
Figure 1: The plasmin system in milk. Modified from Ismail & Nielsen (2010).
The physiological role of plasmin in blood is fibrinolysis, i.e. the solubilisation of fibrin clots, a
precisely regulated process that mainly depends on the binding of the enzymes to their substrate
fibrin (Cesarman-Maus & Hajjar, 2005). In milk, plasmin, plasminogen and tPA are mainly
bound to the casein micelles (Baer, et al., 1994; Benfeldt, Connie, et al., 1995; Haissat, et al.,
1994; Politis, et al., 1992; Politis, et al., 1994; Wang, L., et al., 2006; White, et al., 1995). uPA,
on the other hand, has been shown to be associated with somatic cells by uPA receptors
(Heegaard, Rasmussen, et al., 1994) and upon release from the somatic cells associates with the
casein micelles as well (Heegaard, Rasmussen, et al., 1994; Lu, D. D. & Nielsen, 1993a; White,
et al., 1995). The inhibitors of the plasmin system are located in the serum phase of milk
(Christensen, S., et al., 1995; Precetti, et al., 1997).
10
4.2.2 Molecular features of plasmin and plasminogen
Bovine plasminogen has a calculated molecular mass of 88,092 Da comprising 786 amino acid
residues and is glycosylated at two positions (Marti, et al., 1988; Schaller, et al., 1985).
Plasminogen consists of an N-terminal pre-activation peptide, 5 homologous kringle structures
and a C-terminal catalytic centre (Figure 2). The overall structure is stabilized by disulphide
bonds. The kringle structures contain lysine binding sites with different affinities for lysine, by
which plasminogen and plasmin bind to lysine residues in the casein micelles (Bastian & Brown,
1996; Benfeldt, Connie, et al., 1995). The amino acid composition of human and bovine
plasminogen differs, but the position of the cysteine residues is the same and based on the
sequences the kringles are homologous, indicating their structure is conserved (Bastian & Brown,
1996; Schaller, et al., 1985).
Figure 2: Schematic representation of plasminogen with the pre-activation peptide (PAP), kringle structures (K1-K5) and light chain containing the catalytic center. The cleavage sites and enzymes involved in the activation of plasminogen are indicated in red. Disulphide bonds are shown as dashed lines. Modified from Benfeldt et al. (1995).
11
The crystal structure of human plasminogen kringle 4 in complex with the lysine analogue ε-
aminocaproic acid (EACA) is shown in Figure 3. As can be seen, the ε-amino group of lysine or
EACA is stabilized by two aspartic acid residues. Additionally, the hydrophobic C4 chain is
stabilized by two phenylalanine and one tryptophan residue (Ponting, et al., 1992; Wu, et al.,
1991). The binding of lysine or EACA to the kringle structures further results in a conformational
change of the kringle structures (Wu, et al., 1991). Binding to lysine residues or lysine analogues
also affects the entire plasminogen molecule; from a compact and folded to a looser, unfolded
one (Hayes, K. D., et al., 2003; Mangel, et al., 1990; Markus, 1996). The electrostatic nature of
the lysine binding sites can explain why plasmin is not dissociated from the casein micelles at
low temperatures. On the other hand, addition of 1 M NaCl or at pH values < 4.6 plasmin
dissociates from the casein micelles which is the cause for the higher plasmin activity in acid
whey compared to sweet whey (Crudden & Kelly, 2003; Grufferty & Fox, 1988b; Kelly &
McSweeney, 2003).
Figure 3: The refined structure of the ε-aminocaproic acid complex of human plasminogen kringle 4. The amino acids of plasminogen kringle 4 involved in the complexion of ε-aminocaproic acid are annotated (Wu, et al., 1991).
The catalytic centre of plasmin is located in the C-terminal region and contains the serine
protease specific catalytic triad consisting of His-Asp-Ser located at the amino acid residues 598,
641 and 736, respectively (Hedstrom, 2002; Schaller, et al., 1985).
12
4.2.3 Activation of plasminogen
Plasminogen is activated by conversion from a single chain zymogen to a two chain active
enzyme connected by disulphide bonds by cleavage of the peptide bond Arg557-Ile558 in
plasminogen (Bastian & Brown, 1996). Plasmin is able to cleave plasminogen at Lys77-Arg78
which yields the so called pre-activation peptide. This cleavage results in a conformational
change of plasminogen in which the Arg557-Ile558 bond is exposed. The rate of activation of
plasminogen is therefore increased in the presence of even small amounts of plasmin (Bastian &
Brown, 1996; Cesarman-Maus & Hajjar, 2005).
Both uPA and tPA are serine proteases and both show some homology with plasmin. In the
human system, both activators exist in both a single and a two-chain form, with different
activation efficiency (Cesarman-Maus & Hajjar, 2005). Activation of plasminogen by tPA in
blood is weak in absence of fibrin. The activation of plasminogen by tPA increases by several
orders of magnitude when both are bound to fibrin in blood or casein in milk, respectively
(Cesarman-Maus & Hajjar, 2005; Markus, et al., 1993). Especially the αS2-casein dimer and κ-
casein multimers are shown to accelerate the activation of plasminogen by tPA (Heegaard,
Rasmussen, et al., 1994; Heegaard, et al., 1997). In contrast to tPA, uPA does not bind to fibrin
and the rate of activation by uPA is not affected by the presence of fibrin (Lu, D. D. & Nielsen,
1993a; White, et al., 1995). Casein has been shown to enhance the activation of human
plasminogen by uPA, which is most likely caused by the conformational change in plasminogen
upon occupation of lysine binding sites as described above (Baer, et al., 1994; Markus, et al.,
1993; Politis, et al., 1995). In model studies, both native and denatured α-la and denatured β-lg
stimulated the activation of bovine plasminogen by human uPA. The enhanced rate of
plasminogen activation was suggested to be caused by a faster release of plasmin from the
plasminogen-uPA complex in the presence of whey proteins (Rippel, et al., 2004).
Benfeldt et al. (1995) purified and characterised a low molecular weight plasmin/plasminogen
with a molecular weight of 50 kDa. This so-called midi-plasminogen/midi-plasmin was
hydrolysed between Arg343-Met344 and consisted only of kringle 4 and 5 and the light chain. The
cleavage was suggested to be autocatalytic by plasmin or uPA. This midi plasminogen was
especially found in the serum phase, suggesting that the loss of kringle 1-3 reduced the binding to
caseins. Whether the full or midi form of plasmin and plasminogen are the predominant form in
milk is unknown since the concentration of midi plasminogen could not be determined, but it was
suggested that the midi-plasmin form was the major form of the bovine plasmin molecule in milk
(Benfeldt, Connie, et al., 1995).
13
4.2.4 Inhibitors of plasmin and plasminogen activators
There are at least six inhibitors of plasmin present in milk: α2-antiplasmin, α2-macroglobulin,
antithrombin III, C1-inhibitor, inter-α-trypsin inhibitor and bovine plasma-trypsin inhibitor
(Christensen, S., et al., 1995). The two most important milk inhibitors are the plasmin specific α2-
antiplasmin and the non-specific α2-macroglobulin (Kelly & McSweeney, 2003). The α2-
antiplasmin belongs to the inhibitor family of serpins. All serpins form an irreversible complex
with serine proteases, which is followed by a cleavage of the inhibitor, and both the protease and
inhibitor lose their activities (Cesarman-Maus & Hajjar, 2005; Christensen, S. & Sottrup-Jensen,
1992; Wiman & Collen, 1978). The function of α2-antiplasmin in blood is the inhibition and
inactivation of potentially harmful plasmin being released from fibrin clots. The formation of the
plasmin-α2-antiplasmin complex requires a free lysine binding site on plasmin. Christensen et al.
(1992) found that α2-antiplasmin inhibits both bovine plasmin and midi plasmin, suggesting that
α2-antiplasmin binds to the lysine binding site in kringle 4 of plasmin. The reaction involves a
very fast first step in which α2-antiplasmin binds to plasmin and a slower second step in which
the irreversible complex is formed (Christensen, U., et al., 1996; Wiman & Collen, 1978). Prado
et al. (2006) found a competitive inhibition of plasmin in a plasmin inhibitor extract from milk
containing α2-antiplasmin. Since the inhibitor was added to a mix of plasmin and substrate, the
measured inhibition was most likely only the first step of α2-antiplasmin binding to plasmin.
Based on their molecular weights, plasmin and plasmin inhibitor are present in an equimolar
concentration in fresh milk (Prado, et al., 2006; Precetti, et al., 1997). Two types of fast reacting
plasminogen activator inhibitors (PAI-I and PAI-II) have been identified in milk (Kelly &
McSweeney, 2003).
Besides specific protease inhibitors, plasmin can be inhibited by whey proteins. Both native and
denatured forms of β-lactoglobulin act as inhibitors of plasmin activity towards a synthetic
substrate and casein (Bastian, et al., 1993). Besides β-lg, also α-lactalbumin (α-la) and bovine
serum albumin (BSA) inhibited plasmin as well. The variant β-lg A is shown to have a higher
inhibitory effect than β-lg B (Politis, et al., 1993). This difference is surprising since the two
variants differ only in two amino acid residues. Whether this finding is genuine or an artefact of
the assay still needs verification. The mechanism of inhibition of plasmin by whey proteins,
however, is still unknown. In case of a competitive inhibition of plasmin by β-lg, even small
changes in the amino acid sequence could affect the binding of β-lg to the substrate binding site
and explain the findings of Politis et al (1993), which has been shown on the catalytic efficiency
of human plasmin towards similar peptide sequences (Hervio, et al., 2000). It is important to note
14
that the inhibitory effect of the whey proteins is different when synthetic substrates or casein is
used as a substrate (Hayes, M. G., et al., 2002).
4.2.1 Specificity of plasmin
Plasmin shows the same specificity as trypsin and hydrolyses peptide bonds on the C-terminal
side of lysine and, to a lesser extent, after arginine (Kelly & McSweeney, 2003). The primary
substrate for plasmin in milk is β-casein. Plasmin hydrolyses β-casein primarily at Lys28-Lys29,
Lys105-His106 and Lys107-Glu108 which results in the so-called proteose peptones and γ-caseins,
derived from C- and N-terminal part of the β-casein molecule (Eigel, et al., 1984). The
nomenclature and sequence of these polypeptides are given in Table 2.
Table 2: γ-caseins and proteose peptones (PP) of β-casein by plasmin (Eigel, et al., 1984).
Peptide Sequence
PP 8-fast 1-28
PP 8 slow 29-105, 29-107
PP component 5 1-105, 1-107
γ1-casein 29-209
γ2-casein 106-209
γ3-casein 108-209
15
Cleavage occurs also at Lys113-Tyr114 and Arg183-Asp184 (Kelly & McSweeney, 2003). In Paper
III, we did not find any cleavage by plasmin at Arg183-Asp184 in β-casein, but rather at Lys169-
Ala170 and Lys176-Ala177. Furthermore, the peptides identified in Paper III showed that plasmin
hydrolysed β-casein in UHT milk at Lys29-Ile30, Lys32-Phe33, Lys48-Ile49, Lys97-Ala98 and Lys99-
Glu100 (Figure 4), suggesting that the proteose peptone fraction is more heterogeneous than
previously reported.
Figure 4: Primary structure of the bovine β-casein variant A2 5P with plasmin cleavage sites
described in literature and Paper III indicated by arrows (Kelly & McSweeney, 2003). The cleavage site at Arg183-Asp184 was not found in Paper III and is indicated red.
The storage time after which the peptides were identified and the rate of peptide formation
indicated that the plasmin initially hydrolysed at Lys105-His106 and Lys107-Glu108 and sequentially
continued to hydrolyse β-casein further around these cleavage sites at Lys97-Ala98, Lys99-Glu100
and Lys113-Tyr114 (Paper III). The only potential cleavage site in β-casein, which is hydrolysed
by trypsin, but not plasmin, seems to be Arg202-Gly203 (Bumberger & Belitz, 1993).
16
Plasmin hydrolyses αS2-casein at approximately the same rate as β-casein (Kelly & McSweeney,
2003). The major previously reported cleavage sites include Lys21-Gln22, Lys24-Asn25, Arg114-
Asn115, Lys149-Lys150, Lys150-Thr151, Lys181-Thr182, Lys188-Ala189 and Lys197-Thr198 (Le Bars &
Gripon, 1989; Visser, et al., 1989). By now, several additional cleavage sites have been identified
from peptides occurring in the indigenous peptide profile of milk (Baum, Fedorova, et al., 2013;
Dallas, et al., 2013). In Paper III, apart from known cleavage sites, we identified six additional
plasmin specific cleavage sites in αS2-casein that have not been described before (Figure 5). The
reason for the relatively scarce reports on cleavage sites in the literature for αS2-casein may be
attributed to its relatively low concentration in milk and the dimeric nature of αS2-casein, which
complicates peptide analysis of αS2-casein hydrolysates. The high and varying degree of
phosphorylations on αS2-casein further impedes mass spectrometry analysis of αS2-casein peptides
due to a reduced concentration of identical peptides varying in phosphorylations and the reduced
ionisation and fragmentation ability of phosphorylated peptides (Baum, Ebner, et al., 2013;
Tauzin, et al., 2003).
Figure 5: Primary structure of bovine αS2-casein A 11P with plasmin cleavage sites reported in literature and Paper III are indicated by arrows (Baum, Fedorova, et al., 2013; Dallas, et al., 2013; Le Bars & Gripon, 1989). The novel plasmin cleavage sites in αS2-casein reported in Paper III are indicated as grey arrows.
The activity of plasmin towards αS1-casein is lower compared to β-casein (Andrews &
Alichanidis, 1983; Gazi, et al., 2014), which was also observed in Paper II. The major cleavage
sites found for isolated αS1-casein are Arg22-Phe23, Arg90-Tyr91, Lys102-Lys103, Lys103-Tyr104,
Lys105-Val106, Lys124-Glu125 and Arg151-Gln152 (Le Bars & Gripon, 1993; McSweeney, et al.,
1993). In Paper III, we could not find indications for a cleavage at Arg151-Gln152 (Figure 6),
17
which is most likely due to a difference in the accessibility of cleavage sites on isolated and
micellar casein, respectively. The region of αS1-casein, which includes Arg151-Gln152, is
hydrophobic and could be inaccessible for plasmin in a micellar system. The importance of
hydrophobic domains of the caseins was also shown in Paper III for the N-terminal peptide of
αS1-casein formed by plasmin αS1-casein f(1-22). Relative quantification of peptide
concentrations measured by UV and corrected for their calculated molar absorption coefficient
further showed that Arg22-Phe23 is not a major cleavage site in micellar casein, but that the initial
hydrolysis of αS1-casein is in the region 90-105 and Lys124-Glu125 (Paper III). This is in line with
the findings in Paper II, where αS1-casein f(1-124) and αS1-casein f(125-199) were the two large
polypeptides present early in the storage period.
Figure 6 Primary structure of bovine αS1-casein B 8P with plasmin cleavage sites identified in literature and Paper II are indicated by arrows (Baum, Fedorova, et al., 2013; Le Bars & Gripon, 1993; McSweeney, et al., 1993). Dashed arrows indicate cleavage sites only found for isolated αS1-casein (Le Bars & Gripon, 1993; McSweeney, et al., 1993).
Plasmin has little activity on κ-casein compared to the other caseins. Reports on κ-casein
hydrolysis by plasmin vary and the specificity has not been determined (Bastian & Brown, 1996;
Dalsgaard, et al., 2007; Kelly & McSweeney, 2003). The low activity could be caused by the
presence of carbohydrate moieties on κ-casein and its polymeric nature similar to αS2-casein
(Bastian & Brown, 1996).
Only few studies have looked into plasmin activity on whey proteins. In general, whey proteins
are rather resistant to plasmin hydrolysis, although hydrolysis of BSA, lactoferrin, β-lg and α-la
has been reported (Dalsgaard, et al., 2007; Hayes, M. G., et al., 2002; Kelly & McSweeney,
2003). In the peptidomic study conducted in Paper III, we identified 6 peptides originating from
18
β-lg. The cleavage sites were Lys8-Gly9, Lys71-Thr72, Lys75-Ile76, Lys71-Thr72, Lys83-Ile84 and
Lys91-Val92, which were described previously by (Caessens, et al., 1999). Plasmin hydrolysis of
α-la has been reported in model studies (Dalsgaard, et al., 2008). The resistance of whey proteins
towards hydrolysis is most likely due to their globular structure. The β-lg hydrolysed in the UHT
milk of Paper II and Paper III was partially denatured and thus the structure was altered and
easier to hydrolyse. The low levels of α-la denaturation in the UHT milk could explain the
absence of α-la peptides.
4.2.2 Factors affecting the plasmin system
4.2.2.1 Environmental factors
Activity of the plasmin system in milk can be affected by either changes in the transport of its
components from blood to milk or differences in the activation of plasminogen (Kelly &
McSweeney, 2003). Furthermore, presence of inhibitors of either plasmin or plasminogen
activator inhibitors will affect the net activity of plasmin present. Different breeds of cattle show
variations in plasmin activity with milk from Jersey cows having a lower plasmin activity than
milk from Friesian and Montbeliard cows (Benslimane, et al., 1990; Richardson, B. C., 1983b).
Plasmin activity has also been found to be increased in milk from older cows (Kelly &
McSweeney, 2003).
The stage of lactation can largely affect plasmin activity. Plasmin activity decreases and
plasminogen activity increases in milk from the early lactation phase after calving (Benslimane,
et al., 1990; Kelly & McSweeney, 2003). In late lactation milk, both plasmin, plasminogen and
plasminogen activator activity are increased (Baldi, et al., 1996; Richardson, B. C., 1983b). The
increased levels of plasmin and activation of plasminogen in late lactation milk have been linked
to involution of the mammary gland (Politis, Lachance, et al., 1989; Politis, 1996).
Plasmin, plasminogen and plasminogen activator levels are increased in mastitic milk (Kelly &
McSweeney, 2003). This could be caused by an increased transfer of plasmin system components
from the blood into the milk during mastitis due to compromised barrier between milk and blood
as well as elevated plasminogen activator levels attributed to increased levels of somatic cells
(Heegaard, Christensen, et al., 1994; Heegaard, Rasmussen, et al., 1994; Politis, Ng Kwai Hang,
et al., 1989). In general, there is a positive correlation of plasmin activity with the somatic cell
count (Albenzio, et al., 2004; Kennedy & Kelly, 1997; Politis, Ng Kwai Hang, et al., 1989).
Besides mastitis, bacteria present in milk can affect the plasmin system as well. Proteases from
psychotrophic bacteria are able to release plasmin and plasminogen from the casein micelles into
the serum phase and are capable of inactivating plasmin (Fajardo-Lira & Nielsen, 1998;
Frohbieter, et al., 2005). While most bacteria do not activate plasminogen, a protease from
19
Bacillus polymyxa shows plasminogen activator like activity (Larson, et al., 2006; Nielsen, S. S.,
2002; Verdi & Barbano, 1991). A protease from Pseudomonas fluorescens does not activate
plasmin itself, but is capable of increasing the catalytic efficiency of uPA by converting it into its
two chain form (Guinot-Thomas, et al., 1995).
4.2.2.2 Effect of milk processing on the plasmin system
Plasmin and plasminogen activators can be active during cold storage of milk resulting in
increased proteolysis and levels of plasmin activity (Ismail & Nielsen, 2010; Somers, et al.,
2002). Storage of milk at 5 °C, on the other hand, results in decreased levels of plasmin activity
caused by autolysis of plasmin compared to milk stored at room temperature (Crudden, Fox, et
al., 2005). Autolysis has previously been observed for isolated plasmin fractions and autolysis
can be reduced by addition of lysine analogues or providing a protein network for plasmin (Saint-
Denis, et al., 2001b; Ueshima, et al., 1996). The plasmin concentration used in these studies
however is much higher compared to concentration of plasmin in milk and the casein in milk
provides a suitable protein network to stabilize plasmin against autolysis. Most studies reporting
autolysis of plasmin in milk use raw milk (or pressure treated milk) in their experiments
(Crudden, Fox, et al., 2005; Guinot-Thomas, et al., 1995; Huppertz, et al., 2004). The binding of
plasmin to casein is a reversible process and the decrease in plasmin activity could also be
attributed to irreversible inhibition of released plasmin by plasmin inhibitors. No evidence for
autolysis or irreversible inhibition exists in literature, but could help to understand the dynamics
of the plasmin system during the storage of milk. Identification of either plasmin and
plasminogen fragments or the presence of the 10 kDa peptide originating from the N-terminal
part of α2-antiplasmin upon inhibition/inactivation of plasmin in stored raw milk could solve this
question.
Release of uPA from somatic cells could occur during storage of raw milk and prolonged storage
could result in increased levels of uPA in milk. The effect of milk separation, bactofugation and
spore filtration on uPA release from somatic cells has not been studied yet, but is expected to
significantly affect the levels of uPA in milk.
Heat treatment of milk greatly affects the plasmin system. Plasmin, plasminogen and
plasminogen activators are generally considered as heat stable, while the inhibitors of plasmin
and plasminogen activators are relatively heat labile. Low-pasteurisation conditions (72-75°C for
15 s) do not significantly affect plasmin, plasminogen and plasminogen activators (Ismail &
Nielsen, 2010). In fact, pasteurisation results in an increased plasmin activity due to the
inactivation of plasmin inhibitors and plasminogen activator inhibitors (Prado, et al., 2006;
20
Richardson, B. C., 1983a). Plasminogen activation during cold storage of pasteurized milk is thus
increased compared to raw milk (Lu, R., et al., 2009). Plasmin inhibitors have been shown to
have a higher heat stability than plasminogen activator inhibitors (Prado, et al., 2006).
We examined the effect of heat treatment at 72 °C on plasmin activity in a small experiment as
part of the method development for the plasmin assay (Paper I). Plasmin activity in the milk and
the serum phase decreased slightly (Figure 7). While the recovery rate of added plasmin in the
milk was constant during the heat treatment, the recovery rate in the serum increased by almost
10 % with increasing holding time. The increase in recovered activity most likely represented the
inactivation of plasmin inhibitors during the heat treatment. The change in recovered activity
fitted well to a first order reaction, indicating that this method could be used for a kinetic
description of inactivation of plasmin inhibitors. Since the focus of the PhD project was on UHT
milk, the inactivation of plasmin inhibitors was not pursued further in the project.
Figure 7: Effect of heat treatment at 72 °C for up to 195 s on plasmin activity (A) and recovery rate of added plasmin (B) in skimmed raw milk (□) and the serum phase (●) obtained by ultracentrifugation (10
5 rcf for 60 min) after the heat treatment. Bars indicate standard
deviation, n = 3;
The increased plasmin activity after pasteurisation could also be attributed to the denaturation of
plasminogen (temperature range 50.1-61.7 °C), which increases the activation by uPA (Burbrink
& Hayes, 2006). Inactivation of plasmin and plasminogen during heat treatment mainly occurs
via thiol-disulphide interchange reactions with denatured β-lg (Rollema & Poll, 1986). The
thermal inactivation of plasmin, plasminogen and plasminogen activators will be covered in
detail in section 6.3.2.
21
High pressure homogenisation has been shown to inactivate plasmin and plasminogen (Hayes, M.
G. & Kelly, 2003). On the other side, more recent studies have indicated that these first findings
were an artefact of the plasmin assay used in this study, which included a centrifugation step to
remove the cream (and plasmin attached to it). High pressure homogenisation seems to have only
a little effect on plasmin activity itself at elevated pressure (20 % plasmin inactivation at 200
MPa; Iucci, et al., 2008).
High pressure treatment on the other hand has been shown to significantly affect the plasmin
system. High pressure treatment at room temperature up to 400 MPa does not inactivate plasmin
(García-Risco, et al., 2003; Huppertz, et al., 2004) and treatment at 300-400 MPa even promotes
plasmin activity (Huppertz, et al., 2004). Plasmin activity decreases after high pressure treatments
at 600 MPa. The increase in plasmin activity could be attributed to disruption of casein micelles
and transfer of plasmin system components into the serum phase, while the inactivation of
plasmin at higher pressures could be caused by pressure induced denaturation of β-lg (García-
Risco, et al., 2003; Hinrichs & Rademacher, 2004; Huppertz, et al., 2004; Moatsou, et al., 2008).
Studies on the pressure-temperature stability of isolated plasmin indicated a stabilizing effect of
plasmin at pressures >600 MPa and temperatures <50 °C (Borda, et al., 2004).
Cheeses made from milk concentrated by ultrafiltration have reduced plasmin activity and
reduced rates of proteolysis (Benfeldt, C., 2006). The decrease is partially ascribed to the
retention of whey proteins and inhibitors of plasmin and plasminogen activators during the
ultrafiltration and subsequent inclusion in the cheese curd. The time-temperature conditions and
presence of air could have affected the plasmin system, i.e. the inactivation of plasminogen
activators (Benfeldt, C., 2006). During microfiltration and diafiltration, most plasmin and
plasminogen remain in the retentate and exhibit an increased activity since whey proteins and
inhibitors are removed by the microfiltration (Aaltonen & Ollikainen, 2011). We found similar
trends in a pilot scale extended diafiltration (10 diafiltration steps, 20°C, pasteurised skim milk).
22
Plasmin activity varied during the diafiltration process, which could mostly be due to varying
casein concentrations in the casein retentate (Figure 8A). After correction for casein
concentration, plasmin activity increased by 35-37 % after five diafiltration steps. More than
95 % of whey proteins were removed from the casein retentate after four diafiltration steps. The
plasmin activity in the permeate was much lower compared to the retentate and stayed constant
during the filtration. The recovery rate in the permeate on the other hand decreased with
increasing whey protein concentration, indicating that small amounts of plasmin dissociated form
casein micelles and permeated. The recovery rate in the casein retentate increased rapidly (Figure
8B) until the whey proteins were removed. The further increase in recovery rate could be
attributed to a slower permeation of plasmin inhibitors with a higher molecular weight than whey
proteins. As reported by Aaltonen & Ollikainen (2011), we also found a linear correlation
between whey protein concentration and plasmin activity.
Figure 8: Effect of diafiltration of pasteurized milk on plasmin activity (A) and recovery rate of plasmin activity (B) in the casein/retentate (□) and serum whey/permeate fraction (●). Bars indicate standard deviation, n = 3.
23
5 Measurement of the plasmin system activity
There are several possibilities to measure enzymes in milk. Enzyme concentration is usually
measured by immunoassays such as enzyme-linked immune sorbent assay (ELISA), which are
(in some cases) resistant to inhibitory and interfering compounds. A second possibility are
activity measurements which aim to measure the effective enzyme activity in samples and take
inhibitory effects into account. A third possibility is the measurement of products produced by
the enzyme (Kelly & Fox, 2006). The most optimal method depends on the aim of the
measurement. ELISA gives the concentration of the enzyme, but might not differentiate between
active and inactive enzymes. Activity measurements usually use synthetic substrates and are
largely affected by the buffers in use and the sample preparation. Analysis of enzyme reaction
products might give the most unadulterated view of the effect of enzyme activity in a product, but
the analysis of these products can be complex and, in the case of low enzyme activity, very time
consuming. In the following, different ways of measuring the plasmin system will be presented
and discussed.
5.1 Activity assays
5.1.1 Plasmin and plasminogen
Activity assays for plasmin use a synthetic substrate, i.e. small peptides containing a plasmin
sensitive bond. Upon cleavage, a fluorescent or coloured product is released, which then can be
quantified by measuring fluorescence or light absorbance (Figure 9). Synthetic substrates for
plasmin are N-Suc-Ala-Phe-Lys-7-amido-4-methyl coumarin (fluorescence), D-Val-Leu-Lys-p-
nitroanilide (S-2251, colorimetric) and HD-Norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide
(Spectrozyme-PL, colorimetric).
Figure 9: Schematic representation of the release of yellow p-nitroalinine by cleavage of S-2251 by plasmin.
24
Plasminogen (derived) activity is measured as plasmin activity after activation of plasminogen.
The difference between plasmin and plasmin plus plasminogen derived activity is used to
calculate plasminogen activity.
Besides the different substrates, a variety of buffers and sample preparations are used, which can
affect the outcome of the measurement. It is obvious that there are difficulties in comparing
results obtained by different assays and, maybe even more important, to infer to the
characteristics of the plasmin system in milk from these measurements. This problem was already
addressed by Kelly et al. (2006), who recommended a standardisation and comparison of plasmin
activity assays.
A considerable part of this PhD study dealt with the effect of buffer, sample composition and
preparation on the outcome of plasmin activity measurements. Some of the key results were
published in Paper I.
5.1.1.1 Effect of sample preparation on plasmin activity
Spectrophotometric and fluorescence analyses generally require a clear sample with as little
scattering and quenching as possible. In milk, the main components that interfere with these
measurements are casein micelles and fat. Accordingly, most described assays use sodium citrate
or ethylenediaminetetraacetic acid (EDTA) to dissociate the casein micelles by complexion of
calcium and calcium phosphate on skimmed samples. These additions do not dissociate the casein
micelles completely, but results in small particles with sizes between 15 and 50 nm (McMahon &
Oommen, 2013). Plasmin and plasminogen are most likely still bound to these particles, since the
pH values of sodium citrate and EDTA solutions are usually above pH 4.6. In a preliminary study
in the present work, no significant differences between the treatment with ε-amino caproic acid
(EACA) or sodium citrate on plasmin activity were observed (results not shown), concluding that
both reagents can be used.
A different approach is the incubation of the sample with substrate and the use of Clarifying
Reagent to remove the turbidity before measurement (Saint-Denis, et al., 2001b). Politis et al.
(1993) used EACA to release plasmin and plasminogen from the casein micelles and removed
them by ultracentrifugation, resulting in a clear sample containing only plasmin and plasminogen.
In order to avoid turbidity caused by fat, plasmin analysis is either carried out on skim milk
samples (Rollema, et al., 1983) or includes a centrifugation step to remove the fat (Richardson, B.
C. & Pearce, 1981). Using the assay described in Paper I, we were able to measure plasmin
activity without removal of the fat phase. This was possible by correcting for turbidity using a
second wavelength that represented the background (Prado, et al., 2006). The results showed that
removal of the fat did not affect plasmin activity in unhomogenised raw milk, but the plasmin
25
activity decreased, when the fat was removed from pasteurized, homogenized milk. Upon
homogenisation, the milk fat globule membrane (MFGM) becomes to consist to a considerable
degree of caseins and whey proteins (Cano-Ruiz & Richter, 1997; Kessler, 2002). Removal of the
cream phase thus results in the removal of plasmin associated with caseins on the milk fat
globules. The same effect was observed when the surfactant Tween was added prior to the
removal of fat of homogenized milk (Iucci, et al., 2008). As a result, the decrease in plasmin
activity during high pressure homogenisation was found to be an artefact of the sample
preparation rather than a real inactivation of plasmin (Hayes, M. G. & Kelly, 2003; Iucci, et al.,
2008). This shows how important the consideration of interaction of plasmin, milk components
and milk processing are for the design of an appropriate plasmin activity assay.
In milk samples subjected to extreme heat treatment at 90 °C for 5 -30 min, large protein
aggregates were formed and interfered with the assay presented in Paper I. These aggregates
sedimented during the performance of the assay within 2 h and due to the correction for turbidity,
an apparent increase in absorbance was observed in these samples. Although no plasmin activity
is to be expected in these samples, the use of Tween and centrifugation of the sample would be a
more appropriate sample preparation. At the time of the assay development, we were
unfortunately not aware of the work of Iucci et al. (2008).
5.1.1.2 Effect of sample composition and buffers on plasmin activity
In addition to the previously discussed inhibition of plasmin by whey proteins and plasmin
inhibitors, the plasmin activity in an assay is also affected by the caseins. Caseins are the natural
substrate for plasmin in milk and thus compete with the synthetic substrate for the active site of
plasmin. They have been shown to act as competitive inhibitors against the synthetic substrate,
which can be overcome by increasing the substrate concentration (Bastian, et al., 1991). The state
of caseins (monomeric, small aggregates or intact micelles) might affect the interfering effect due
to the differences in their mobility, diffusion rate and accessibility. Saint-Denis et al. (2001a)
conclude that the main inhibitory components in the plasmin assay are the caseins. This is based
on a linear decrease of plasmin activity in a dilution of pasteurized milk (containing native whey
proteins and inhibitors) with UHT milk (denatured whey proteins, no inhibitors) compared to a
non-linear decrease of plasmin activity, when pasteurized milk is diluted with buffer, similar to
the findings reported in Paper I. Our results in Paper I suggest that the whey proteins are the
main inhibitory components and that the difference in inhibition by native and denatured whey
protein might not be significant, when analysing milk samples. Additionally, both native and
denatured whey proteins have been shown to inhibit plasmin activity (Bastian, et al., 1993;
Politis, et al., 1993). This together with our results
26
More recent studies indicate that the inhibitory effect of whey proteins on plasmin activity is
affected by the choice of substrate, as some substrates may interact with the whey proteins
(Rippel, et al., 2004). The substrate, S-2251, is shown to be able to bind to whey proteins by
hydrophobic interactions, while another substrate for plasmin activity, Spectrozyme-PL, is not
affected by whey proteins due to its more hydrophilic nature (Rippel, et al., 2004). Using the
fluorescent substrate N-Suc-Ala-Phe-Lys-7-amido-4-methyl coumarin, the whey proteins have
either an inhibitory or promoting effect at pH 6.5 or pH 5.2, respectively (Hayes, M. G., et al.,
2002). These studies were carried out in model systems with plasmin and isolated whey proteins.
For the milk and casein system used in our study, it could be expected that the casein particles,
which have considerable hydrophobic domains, would be able to adsorb the substrate S-2251 due
to their higher concentration. However, the dilution of a casein solution with buffer in Paper I
did not show a significant concentration dependent decrease in plasmin activity, suggesting that
the inhibitory effects measured in our study reflect real inhibition rather than substrate binding of
whey proteins.
Figure 10: Measured activity of added plasmin to skimmed raw milk (○), pasteurized skim milk (●) and UHT milk (□). Activity of added plasmin was measured in the assay buffer described in Paper I containing gelatine to stabilize plasmin. Error bars indicate standard deviation, n = 3.
Apart from these factors, the buffers used for plasmin analysis have a large impact on the results.
Buffers for plasmin analysis often contain NaCl and lysine derivatives like EACA to dissociate
plasmin from the caseins and to enhance plasmin activity (Bastian, et al., 1991). We showed in
27
Paper I that dissociation of plasmin from caseins resulted in a decreased interference from the
caseins in the assay, but on the other hand increased the inhibition of plasmin by inhibitory
components. Since EACA reduces the inhibition of plasmin by α2-antiplasmin, the major
inhibitory components were most likely the whey proteins. An inhibitory effect of plasmin
inhibitors cannot be completely excluded, as can be seen in Figure 10. Addition of plasmin to
different milk samples showed that for raw and pasteurized milk, a certain amount of plasmin
activity disappeared, when added in low concentrations, while a constant inhibition/interference
was found for UHT milk (Figure 10). This disappearance could be caused by
inhibition/inactivation of added plasmin by plasmin inhibitors.
5.1.1.3 Comparison of activity assays
Table 3 gives an overview of plasmin and plasminogen derived activity assays described and
used in literature. The early plasmin assay by Richardson & Pierce (1981) and by Rollema et al.
(1983) uses a simple sample preparation, i.e. dissociation of caseins and addition of substrate in a
buffer. The methods differ only by the used substrate, N-Suc-Ala-Phe-Lys-7-amido-4-methyl
coumarin and S-2251 in the assays described by Richardson & Pearce (1981) and Rollema et al.
(1983), respectively, and dissociation of plasmin from caseins EACA in the assay used by
Rollema et al (1983). The fluorescence assay by Richardson & Pearce (1981) shows a higher
sensitivity and better level of detection compared to other colorimetric assays. Fluorescence
assays, on the other hand, require a more careful sample handling (i.e. light exposure, differences
in pH and turbidity). Both assay types require a skimming of the sample and are affected by
inhibitors of plasmin and interference from caseins.
28
Table 3: Overview of different plasmin and plasminogen derived activity assays described in literature. If not indicated otherwise, the sample preparation refers to milk samples.
Analysis Measurement Buffer Sample preparation Reference
Plasmin,
plasminogen
Fluorescence
Ex. 380 nm,
Em. 460 nm
0.4 M sodium
citrate
Dissociation of caseins by sodium citrate
Centrifugation/skimming to obtain clear
sample
Mixing with buffer and addition of
substrate
Richardson
& Pearce
(1981)
Plasmin,
plasminogen
Spectrometric,
Abs. 405 nm
40 mM Tris-HCl,
pH 7.4, 100 mM
EDTA, 50 mM
EACA
Addition of skimmed milk to buffer
containing substrate
Rollema et
al. (1983)
Plasmin Spectrometric,
Abs. 405 nm
50 mM Tris, 110
mM NaCl, 3 mM
EACA, pH 7.4
Model study
Mixing plasmin with buffer and reagents
Bastian et
al. (1991)
Plasmin plus
plasminogen
Spectrometric,
Abs. 405 nm
50 mM Tris-HCl,
pH 8, 110 mM
NaCl, 50 mM
EACA
Centrifugation to obtain casein/plasmin
pellet
Resuspending pellet in extraction buffer
Centrifugation to remove caseins
Mixing with substrate buffer
Politis et
al. (1993)
Plasmin,
plasminogen
Fluorescence,
Ex. 370 nm,
Em. 440 nm
100 mM Tris-
HCl, pH 8, 0.4 M
NaCl, 8 mM
EACA
Mixing of sample with extraction buffer
(EACA and NaCl)
Mixing of sample with substrate buffer
Removal of turbidity and stopping of
enzymatic reaction by Clarifying Reagent
No information on fat content of milk
Saint-
Denis et al.
(2001a)
Plasmin Spectrometric,
Abs. 405 nm
Abs. 490 nm
50 mM Tris, 0.1
M NaCl, Tween
80, pH 7.6
Model study
Mixing plasmin with buffer and reagents
Correction for turbidity
Prado et al.
(2006)
Plasmin Fluorescence
Ex. 380 nm,
Em. 460 nm
0.4 M sodium
citrate
Tween
Dissociation of caseins by sodium citrate
Centrifugation/skimming to obtain clear
sample in presence of Tween
Iucci et al
(2008)
Plasmin,
plasminogen
Spectrometric,
Abs. 405 nm
Abs. 490 nm
0.4 M sodium
citrate, pH 9; 100
mM Tris-HCl, pH
8, 0.4 M NaCl, 8
mM EACA
Dissociation of caseins by sodium citrate
Dissociation of caseins and plasmin by
EACA and NaCl
Addition of substrate
Paper I
29
Politis et al. (1993) proposed an assay in which both inhibition and interference are eliminated.
The assay includes a first ultracentrifugation step to remove serum based inhibitory components.
The casein pellet is treated with EACA and NaCl to release plasmin, which is separated from the
caseins in a second ultracentrifugation step before plasmin activity is measured. By this, plasmin
and plasminogen derived activity can be measured without the interference and inhibition of milk
components. The assay is very time consuming and it is questionable, if the assay is applicable
for different sample matrices and processed products. Furthermore, the number of sample
preparation steps will introduce sources of variation.
The highest sensitivity and lowest level of detection is achieved by Saint-Denis et al. (2001)
using high substrate and sample concentrations. Plasmin is dissociated from the casein micelles
and incubated with the substrate. Turbidity is removed by addition of Clarifying reagent. The
drawback of this assay is the point wise measurement compared to a continuous measurement
used in the assays previously described. The increase in fluorescence is measured at three time
points, which increases the number of measurements, time intensity and a poorer precision of the
increase in fluorescence. Saint-Denis et al. (2001) also propose a correction factor for the
interference of caseins referring to apparent and real activity. Since the whey proteins also do
have an impact on plasmin activity, this recovery rate correction includes not only the
interference by caseins, but also inhibitory effects. Since the inhibition and interference cannot be
distinguished, the term recovery rate may be more correct than real activity.
The assay used by Prado et al. (2006) and Rippel et al. (2004) is used in model studies, but
includes a second wavelength measurement to correct for turbidity, which increases the
sensitivity and LOD of colorimetric assays compared to Rollema et al. (1983).
Iucci et al. (2008) use Tween to recover plasmin associated to fat globules in homogenized milk
samples, which enables the measurement of plasmin activity in homogenized samples.
The assay presented in Paper I allows for measurement of plasmin activity in turbid samples and
does not require a centrifugation step. This enables analysis of plasmin activity in different dairy
products and processed milk samples.
All the described assays measure plasmin activity, but the term itself is ill defined. It is used to
describe the concentration of active enzyme in a sample (Politis, et al., 1993), the activity
corrected for interfering effects of caseins (Bastian, et al., 1991; Saint-Denis, et al., 2001b), or the
activity of plasmin including interfering and inhibitory effects (Richardson, B. C. & Pearce,
1981; Rollema, et al., 1983). Due to the complexity of the plasmin system and the changes during
activity assays (dissociation of casein micelle, release of plasmin, removal of components),
conclusions on plasmin in milk based on activity measurement should be considered carefully.
30
5.1.2 Plasminogen activators
Plasminogen activator assays are based on the addition of plasminogen and the resulting increase
in plasmin activity due to activation of plasmin by plasminogen activators. Both fibrin and
synthetic substrates for plasmin have been used in literature to measure plasminogen activator
activity (Korycha-Dahl, et al., 1983; Lu, D. D. & Nielsen, 1993c). Most assays use plasminogen
activator fractions isolated from milk. This has the advantage that a differentiation between uPA
and tPA is possible. uPA, but not tPA is inhibited by amiloride, while tPA activity on the other
hand is extremely low in the absence of fibrin or presence of EACA (Lu, D. D. & Nielsen, 1993c;
Vassalli & Belin, 1987; White, et al., 1995). Saint-Denis et al. (2001) describe an assay to
measure plasminogen activator activity directly in milk, but no differentiation between uPA and
tPA is possible. Compared to the analysis of extracts, the assay by Saint-Denis et al. (2001) is
closer to the actual situation in milk. Addition of amiloride to the assay could allow for a
distinction between tPA and uPA. It is however unclear, if the caseins could enhance tPA
sufficiently in milk to measure tPA activity (Prado, et al., 2007).
Model studies with human plasminogen showed that synthetic substrates containing lysine
residues can promote plasminogen activation by uPA. At the same time, the substrate can bind to
lysine binding sites on plasminogen, resulting in a conformational change in plasminogen and
releasing plasminogen from a protein matrix. This would greatly decrease activation of
plasminogen by tPA, (Kolev, et al., 1995).
Plasminogen of various origins (human, bovine or ovine) is used for plasminogen activator
assays, making a comparison between different assays almost impossible. Additionally, the
available plasminogen preparations are composed of mixtures of different forms and purities
(plasminogen with or without preactivation peptide, preparations containing EACA or lysine).
The heterogeneity of bovine plasminogen (midi or full) and the potential effect on activation
kinetics of bovine plasminogen has not been investigated yet and should be subject of future
studies.
5.2 Immunoassays
ELISA with mono- and polyclonal antibodies has been used to quantify plasminogen and plasmin
(Baer, et al., 1994; Benfeldt, Connie, et al., 1995; Dupont, et al., 1997; Haissat, et al., 1994;
Politis, et al., 1992). Differentiation between plasmin and plasminogen was achieved by using an
antibody specific for plasminogen and plasmin and an antibody specific for plasminogen only,
although a complete quantification was not possible (Dupont, et al., 1997). Benfeldt et al. (1995)
report a poor response of midi plasminogen using ELISA.
31
ELISA and western blotting have also successfully been used for the identification of plasmin
inhibitors in milk (Christensen, S., et al., 1995; Precetti, et al., 1997).
5.3 Other assays
A special form of activity assay used a ferrocenyl peptide with a plasmin sensitive bond bound to
a gold electrode (Ohtsuka, et al., 2009). Cleavage of the peptide by plasmin resulted in a redox
signal, which could enable the direct measurement of plasmin in a sample.
Fourier-transform infrared spectroscopy has been used to differentiate plasmin and plasminogen
(Ozen, et al., 2003). Subtraction of caseinate and whey protein spectra coupled with multivariate
data analysis allows for quantification of both plasmin and plasminogen in the presence of these
proteins. The lowest concentration of the enzymes in the calibration range is 1000-fold higher
than the natural concentration in milk, limiting its application for dairy products.
32
5.4 Proteolysis
Plasmin activity can also be measured by its action on the caseins. Measurement of proteolysis
gives a real measurement of plasmin activity in milk, but underlying mechanisms such as
activation of plasminogen and inhibitory effects can often not be differentiated. Furthermore,
generation of proteolysis products is also influenced by substrate folding, accessibility and
substrate concentration.
5.4.1 Hydrolysis of caseins
Plasmin activity is usually measured by the hydrolysis of β-casein. Proteolysis can be measured
by different techniques. The most commonly used techniques include gel electrophoresis and
liquid chromatography. Gel electrophoresis has the advantage that the caseins and its larger
cleavage products are separated very well, but it is mainly used for qualitative purposes.
Compared to liquid chromatography, the analysis is more time intensive and shows a lower
dynamic range (Chove, et al., 2011). Reverse phase HPLC coupled with MS was used for the
analysis of casein hydrolysis in Paper II. Figure 11 shows a comparison of UHT milk protein
composition after 0 and 12 weeks of storage from the present study.
Figure 11: Reverse phase HPLC chromatogram of UHT milk with plasmin activity after 0 (bottom) and 12 weeks (top) of storage. Caseins (cn) and plasmin hydrolysis products are indicated. A detailed description for the HPLC method is given in Paper II.
After 12 weeks of storage, almost no residual intact β- and αS1-casein was present in the UHT
milk with plasmin activity. Quantification of casein hydrolysis was only possible for β-casein A1
and αS1-casein 9P due to substantial overlap of the proteolysis products with the other variants
and αS2-casein; a complication also reported by Gazi et al. (2014). The use of the MS signal for
the quantification of intact casein is impaired by day-to-day variation of the signal intensity,
33
limited linearity of the signal, potential ion suppression of overlaying peaks and casein
modification during storage (Paper IV). Capillary electrophoresis has been used for the analysis
of caseins, but has the same limitation in terms of overlay of eluting proteins (Recio, et al., 1996).
5.4.2 Peptide formation
Instead of measuring the hydrolysis of intact caseins, proteolysis products can also be used to
measure plasmin activity. Measurement of peptide formation can be divided into unspecific and
specific methods. Unspecific methods include e.g. measurement of free amino terminals, like
fluorescamine assay or the OPA (o-phthaldialdehyde) method (Nielsen, P. M., et al., 2001), and
the measurement of pH 4.6 or TCA soluble material. Specific methods determine the
concentration of plasmin specific proteolysis products.
Unspecific methods and hydrolysis of caseins can be used for analysis of plasmin activity, when
the presence of other proteases can be ruled out. In many dairy products with active plasmin,
other proteases are likely to be present as well. In cheese, chymosin and peptidases from starter
cultures are present, while in UHT milk proteases from psychotrophic bacteria can be present. In
these cases, plasmin specific peptides should be chosen to evaluate plasmin activity.
Figure 12: Reverse phase HPLC chromatogram of UHT milk with plasmin activity after 0 (bottom) and 12 (top) wk of storage. Some plasmin specific proteolysis products are indicated. A detailed description for the HPLC method is given in Paper II.
Most plasmin proteolysis products are precipitated in trichloroacetic acid (TCA) extracts, while
they are soluble at pH 4.6 (Datta & Deeth, 2003). The main plasmin hydrolysis products found in
pH 4.6 soluble extracts are the proteose peptones (Figure 12). In contrast, γ-caseins associate with
intact caseins and precipitate at pH 4.6. Quantification of the large proteose peptones β-casein
f(1-105) and f(1-107) is difficult due to their similar retention time and overlay of different
34
genetic variants. Additionally, these proteose peptones are further hydrolysed by plasmin. The β-
casein f(1-28) is formed fast and does not show genetic variation compared to β-casein f(1-105)
and f(1-107). The presence of β-casein f(1-25) in Paper III indicates that β-casein f(1-28) can be
further hydrolysed, although at a relatively low rate. Recent studies suggest that αS2-casein f(1-
21) and f(1-24) are suitable peptides to evaluate plasmin activity in milk (Cattaneo, et al., 2014).
Both peptides are formed fast by plasmin, but αS2-casein f(1-24) is rapidly hydrolysed to f(1-21)
(Paper III). Thus, αS2-casein f(1-21) is maybe the most suitable peptide for the evaluation of
plasmin activity in milk, since it is formed quickly and is not hydrolysed further by plasmin.
5.4.3 Kinetic aspects of proteolysis during storage
Measurement of plasmin activity in an assay is relatively easy. Simple hydrolysis usually follows
first order reaction kinetics and the substrate is present in a high concentration, which ensures that
only the linear part of the kinetic is measured during the assay. The assay time is relatively short
(from 10 min to 3 h); hence, changes in activity due to activation of plasminogen are
insignificant. When following plasmin activity and proteolysis during storage of UHT milk
activation of plasminogen, substrate affinity and substrate depletion need to be taken into account
for a kinetic evaluation.
It was initially aimed for that the UHT milks used for Paper II-IV would contain four different
plasmin activity levels, which would enable a comparison and correlation of plasmin activity
measured by the plasmin activity assay of Paper I with proteolysis. A combination of the two
analytical methods would allow for a reliable prediction of plasmin activity and proteolysis in
UHT milk during storage. Unfortunately, the experiment resulted in two UHT milks without
measurable plasmin activity and two UHT milks with identical plasmin activities. Kinetic
modelling of proteolysis and plasmin activity in milk with different plasmin activities was
therefore not possible. The UHT milk with plasmin activity used in Paper II and III permitted a
basic comparison and modelling of proteolysis and plasmin activity in this specific milk.
35
Figure 13: Plasmin (●) and plasminogen derived (○) activity during storage of Milk72 from Paper II. Lines represent first order kinetic fits. Error bars indicate standard deviation, n = 3.
As discussed in Paper II, plasmin activity increased during storage while plasminogen derived
activity decreased. Both the increase in plasmin and decrease of plasminogen could be fitted well
to a first order kinetic model (Figure 13).
The kinetic parameters for plasmin increase and plasminogen decrease were not identical. The
rate of plasminogen decrease was higher than the increase in plasmin activity. As can be seen
from Figure 13, all plasminogen was activated after approximately six weeks of storage. The
kinetic fit for the increase in plasmin activity therefore most likely also included changes in the
inhibition/interference factors in the assay, such as a decreased level of intact caseins.
36
The decrease in β-casein (Paper II) and increase in β- and αS2-casein N-terminal peptides (Paper
III) showed a distinct sigmoidal shape. Figure 14 reveals the decrease of intact β-casein and the
development of β-casein f(1-28) during storage.
Figure 14: Development of β-casein f(1-28) (A) and decrease of intact β-casein (B) during storage. Lines represent kinetic fits using a polynomial and first order reaction kinetic.
The sigmoidal shape originates from the activation of plasminogen and thus increased rate of
proteolysis in the initial part of the storage period and a decrease in the rate of proteolysis during
the final part of the storage period due to substrate depletion. The kinetic of peptide formation or
hydrolysis of caseins should therefore include an additional factor to represent the change in
enzyme concentration. The activation of plasminogen follows a first order reaction kinetic. A
normal first order reaction kinetic for product formation is:
(1)
In the case of plasminogen activation, the rate constant k itself changes in a first order reaction as
well (Figure 14) and could be described as:
(2)
kmax represents the maximum velocity of the reaction, similar to Cmax in (1), and is dependent on
the rate of plasminogen activation depicted as kact. This results in complex reaction kinetics,
which are almost impossible to model due to the presence of two exponential functions.
Plasminogen activator assays use high amounts of added plasminogen, which allows a kinetic
interpretation of plasminogen activation using a polynomial expression rather than a first order
kinetic as indicated in Figure 15:
(3)
The factor a in equation (3) represents the change in reaction rate due to the activation of
plasminogen, while factor b represent the initial plasmin activity present in the sample. The
37
proteolysis could therefore be described by using equation (3) for the initial proteolysis and
equation (1) for the remaining storage time (Figure 14).
A different approach is to assume a linear kinetic for proteolysis in combination with a first order
kinetic to include the increase in plasmin activity:
(4)
Both peptide formation and casein hydrolysis can be described by equation (4) by changing the
prefix of k. Applying this fit allows an accurate description of the reaction until the substrate
depletion phenomenon appears (Figure 15).
Figure 15: Development of β-casein f(1-28) (A) and decrease of intact β-casein (B) during storage. Lines represent kinetic fits using equation (4).
This approach might be more suitable for prediction purposes since the quality of the milk would
be unacceptable when substrate depletion occurs. Bitterness and gelation occurred after 70 % and
> 90 % of β-casein, respectively, was hydrolysed (Paper II). When sufficient kinetic data is
provided, a correlation of the plasmin and plasminogen derived activity measured by the activity
assay with the kinetic of proteolysis could be established, allowing the prediction of plasmin
activity and proteolysis in UHT milk.
The kinetics of αS1-casein hydrolysis and the αS1-casein N-terminal peptide f(1-22) in Paper II
and III showed a different kinetic behaviour compared to β-casein and β-casein f(1-28)
respectively. The decrease in intact αS1-casein in the UHT milk showed an extended “lag” phase
of approximately two weeks. This could have been caused by a reduced affinity and accessibility
of αS1-casein for plasmin. A similar reduced affinity was seen for αS1-casein f(1-22), but in
comparison to αS1-casein f(106-124), the lag phase was identical even though αS1-casein f(106-
124) requires two cleavages by plasmin to be formed. This suggests that plasmin has a reduced
affinity for the cleavage at Arg22-Phe23 and a different peptide might be more suitable for the
38
modelling of the proteolysis. The C-terminal peptide αS1-casein f(194-199) was formed slowly
and was only present in low concentrations and could not be used for modelling either. The
peptide αS1-casein f(106-124) would thus be most suitable to be used for modelling, but the
precursor formation would have to be included into the kinetics, resulting in one or two additional
variables, hence making the modelling complex.
The decrease in intact αS2-casein could not be assessed due to overlap with proteose peptones
eluting at the same time (Paper II) and a quantitative assessment using the MS signal is impaired
by the heterogeneity in phosphorylations of αS2-casein. As discussed before, the N-terminal
peptides of αS2-casein formed by plasmin hydrolysis are present in high concentrations and αS2-
casein f(1-21) can be used for modelling.
39
6 UHT milk
6.1 Heat treatment of milk
The main aim of heat treatment of milk is to partially or fully inactivate microorganisms and
enzymes to guarantee consumer safety and prolong the shelf life of milk. Increasing the time
and/or temperature of the heat treatment results in increased inactivation of microorganisms and
enzymes, but can negatively affect other milk constituents. Heat treatment of milk is therefore
often a balance between inactivation of undesired components and preservation of desired
properties.
Heat treatments can be categorized by their time temperature combinations, which are defined by
norms and legislation. Pasteurisation can either be high temperature, short time (HTST) with
heating at 72-75 °C for 15-30 s, or low temperature, long time as e.g. thermisation at 62-65 °C for
30 min. Pasteurisation results in the inactivation of pathogen microorganisms and gives a
negative result when tested for alkaline phosphatase. Any other time temperature combination
resulting in the same effect can be applied as well. Milk subjected to high temperature
pasteurisation is heat treated at temperatures > 85 °C and should give lactoperoxidase negative
test results (Kessler, 2002). Ultra high temperature (UHT) treatment is defined as high
temperature (>135 °C) for a short time, usually 135-150 °C for 1-10 s. The aim of UHT treatment
is to achieve commercial sterility, i.e. free from microorganisms that can grow under storage
conditions (EC, 2005). The effect of UHT treatment on relevant milk constituents will be
discussed later in this section.
6.2 UHT milk processing
6.2.1 Design of UHT processes
The inactivation rate of microorganisms at a given temperature is usually measured by the
decimal reduction time, D, which is the time necessary to reduce the number of microorganisms
by a factor of 10. The temperature effect of the inactivation of microorganisms can be expressed
by the z value and the Q10 value. The z value is defined as the change in temperature necessary to
increase D by a factor of 10. The Q10 value on the other hand is defined as the change in the
inactivation rate by a temperature increase of 10 °C (Kessler, 2002). Chemical effects occurring
during heat treatment can be described using similar parameters as described above. Based on
these relationships, lines of equal effects, i.e. time temperature combinations resulting in the same
inactivation of microorganisms or chemical changes can be defined to design UHT processes.
40
Kessler & Horak (1981) introduced the biological effect B* and the chemical effect C* to
determine time temperature regions for UHT milk processing. A B* value of one equals the 9
decimal reduction of natural thermophilic spores, which results in a commercial sterile product
(Kessler & Horak, 1981; Kessler, 2002). The negative effect of heat treatment on milk
constituents can be characterized by the chemical effect or C* value. A C* value of one equals
the chemical effect of boiling the product for 1 min. Similar to the B* value, a C* value can be
used to define the acceptable limit of chemical changes. In milk, this limit correlates with a loss
of 3 % thiamine and corresponds to a C* value of one (Kessler, 2002). Based on these two
factors, the optimal time temperature conditions for UHT processes are in an area where B* > 1
and C* < 1 (Figure 16).
In the last decade, UHT processes with ultra-short holding times have been developed that are
beyond these time temperature regimes concerning the holding time. It has been shown that UHT
treatment > 150 °C with holding times of 0.2 s result in a sufficient spore reduction to obtain a
commercially sterile product (Huijs, et al., 2004; van Asselt, et al., 2008). The UHT process used
in Paper II-IV (> 150 °C, < 0.2 s) has been validated by Arla on full scale and gave a
commercially sterile product.
Figure 16: Bacteriological B* and chemical C* effect and UHT processing area. Adapted from Kessler (2002).
41
6.2.2 UHT processing systems
UHT processing systems can be divided into direct and indirect systems. In indirect systems, the
product is heated by counter flow of cold milk and hot water or steam in a tubular or plate heat
exchanger. In direct systems, the product is heated by directly mixing it with steam under
pressure, which is later removed using vacuum cooling (Burton, 1994a; Kessler, 2002). A
comparison of a direct, indirect and the direct UHT process used in the trials described in Paper
II-IV is shown in Figure 17.
Figure 17: Schematic representation of time temperature profiles of indirect (dotted line), direct (dashed line) and direct/ultra-short time (solid) UHT processes. Holding time prior (indirect) and after (direct) UHT treatment represent the homogenisation step.
As can be seen from Figure 17, a large advantage of direct systems is the very fast heating rate,
which is only a fraction of a second and that results in minimal chemical changes to the product
compared to indirect systems. One of the disadvantages of using steam to directly heat the
product are that high quality steam is required, and so far the energy recovery in the process is
poor. The product is therefore usually heated to 60-80 °C by indirect heat exchangers. While in
indirect systems, the energy recovery is approximately 90 %, it is only 45 % when using direct
systems, making direct UHT systems costlier to operate (Burton, 1994a; Kessler, 2002;
Malmgren, 2007).
The homogenisation step can be placed before or after the UHT treatment in indirect systems,
while it is always placed after the UHT treatment in direct systems to avoid the formation of
aggregates (Burton, 1994a; Datta, et al., 2002).
42
Direct UHT systems can be further distinguished into steam injection and steam infusion systems
(Figure 18). In steam injection systems, the steam is “injected” as small bubbles into the product.
In steam infusion systems, the product falls as a thin stream or film through a steam pressured
chamber. Steam infusion is generally considered to be gentler than steam injection. During steam
injection, steam bubbles condensate and collapse leading to cavitation and a homogenisation
effect, whereas the steam condensates on the surface of the falling product in steam infusion
systems. A homogenisation effect can be observed in direct steam infusion systems as well, most
likely caused by cavitation occurring during the vacuum cooling (Burton, 1994a; Datta, et al.,
2002; Hougaard, et al., 2009). Fouling is reduced in steam infusions systems since in contrast to
steam injection systems the product does not come into contact with surfaces hotter than itself
(Datta, et al., 2002).
Figure 18: Tetra Pak steam injector nozzle (left) and APV steam infusion chamber (right). Pictures from www.tetrapak.com and www.apv.com.
The UHT trials described in Paper II-IV were performed in the UHT pilot plant (APV/SPX,
Silkeborg, Denmark) at the Arla Strategic Innovation Center in Stockholm. The pilot plant
(Figure 19) is able to run indirect, steam infusion and steam injection UHT processes with a
capacity of 150 l/h. Both tubular and plate heat exchangers can be used to heat or cool the
product. Holding times can be controlled by an external holding cell (15-300 s). The pilot plant is
equipped with a two-stage homogenizer with a maximum pressure of 40 MPa. In the direct UHT
process, the temperature for the flash vacuum cooling was set to 8 °C lower than the pre-heat
temperature to obtain the same dry matter content. Bottling of the milk samples was done in a
sterile bench and every batch was checked for sterility according to (ISO-4833, 2003).
43
Figure 19: The UHT pilot plant at the Arla Strategic Innovation Center Stockholm.
6.3 Heat-induced changes in milk
6.3.1 Proteins
6.3.1.1 Whey proteins
The whey proteins are a heterogeneous group of proteins that are found in the serum phase after
precipitation of caseins by acidification or renneting. The effect of heat treatment on the two
major whey proteins, β-lg and α-la, has been researched intensively in milk and isolated proteins
under different conditions.
When looking at the denaturation of whey proteins, a distinction between reversible and
irreversible denaturation has to be made. Differential scanning calorimetry measurements show
that β-lg and α-la unfold and lose their tertiary globular structure at 75-80 °C and 65 °C,
respectively (Paulsson, et al., 1985; Tolkach, 2007). This denaturation of the whey proteins is
highly reversible. β-lg contains five cysteine residues of which four form disulphide bridges and
one free cysteine residue. In its native form, the free cysteine residue is buried in the globular
structure of β-lg. Upon unfolding of β-lg at higher temperatures (> 75-80 °C), the free cysteine
residue and hydrophobic residues become exposed and are able to react via thiol-thiol, thiol-
disulphide and hydrophobic interactions with other proteins; as a result, b-lg denatures
irreversibly and forms aggregates (Anema, 2008; Sawyer, 2003). Isolated, α-la is very resistant to
irreversible heat induced denaturation (Wang, Q., et al., 2006). However, it contains 4 disulphide
44
bonds and the irreversible denaturation is accelerated in the presence of β-lg (Anema, 2008;
Tolkach, 2007).
The kinetics of irreversible heat induced denaturation of α-la and β-lg consist of two reactions
with different activation energies. The Arrhenius plots for α-la and β-lg show a distinct
discontinuity at 80 and 90 °C, respectively. Below 80 and 90 °C, the activation energies are high,
indicating that a large number of bonds are broken and the rate limiting factor for irreversible
denaturation is the unfolding of the whey proteins. Above 80 and 90 °C, the activation energy is
much lower and the whey proteins are completely unfolded and the speed limiting factor is the
aggregation of the whey proteins (Anema, 2008; Dannenberg & Kessler, 1988; Tolkach, 2007).
Besides the formation of aggregates between whey proteins, β-lg can react with κ-casein on the
surface of the casein micelle via thiol-disulphide interchange reactions (Anema, 2007).
Irreversibly denatured whey proteins precipitate at pH 4.6 and the degree of denaturation of whey
proteins is usually measured as the concentration of pH 4.6 soluble whey proteins before and
after heat treatment. While whey protein denaturation is almost negligible during HTST
pasteurisation at 72 °C for 15 s, UHT treatments can result in almost complete denaturation of
whey proteins. β-lg denaturation is lower in UHT milk processed with direct systems (35-80 %)
than indirect systems (79-100 %) due to the faster heating rate in direct systems (Burton, 1994a;
Datta, et al., 2002). Association of whey proteins with the casein micelles is lower in direct
systems compared to indirect systems. This can also be attributed to the fast heating rate, i.e.
short holding time. Basically, all whey proteins are instantly unfolded during direct UHT
processing, giving the reactive monomers a greater opportunity to react with themselves than
associating with the casein micelles (Anema, 2008).
The whey protein denaturation in the UHT milks produced for Paper II-IV is shown in Table 4.
At a pre-heat temperature of 72 °C, the direct steam infusion step alone resulted in 34 % β-lg and
less than 10 % α-la denaturation. Prolonging the pre-heat treatment to 180 s at 72 °C increased
the degree of whey protein denaturation only slightly. However, increasing the pre-heat
temperature to 95 °C drastically increased the degree of whey protein denaturation. Analysis of
the process showed that the main reason for the increase was caused by the difference in flash
cooling temperature (8 °C lower than the pre-heat temperature) and the higher temperature in the
following homogenisation step, resulting in an increased heat load (approximately 87 °C for 15-
20 s). In the indirect UHT milk and the direct UHT milk 95°C/180 s + > 150°C /< 0.2 s, β-lg is
almost completely denatured (Table 4). There is, however, a significant difference in the α-la
denaturation, indicating that the heat damage was still lower in the direct UHT compared to the
indirect.
45
Table 4: Whey protein denaturation in direct (pre-heat treatment + direct steam infusion step) and indirect UHT milk used for Paper II-IV. Whey protein denaturation was measured as the difference in pH 4.6 soluble whey proteins before and after heat treatment. Values for β-lg are an average of β-lg A and B. Different superscripts indicate significant differences (P < 0.05).
Heat treatment Whey protein
denaturation [%]
β-lg α-la
72°C/5 s + > 150°C /< 0.2 s 33.9 ± 2.7a 9.4 ± 3.2
a
72°C/180 s + > 150°C /< 0.2 s 36.7 ± 3.2a 13.1 ± 3.2
a
95°C/5 s + > 150°C /< 0.2 s 86.6 ± 2.1b 26.6 ± 4.0
b
95°C/180 s + > 150°C /< 0.2 s 94.1 ± 1.6c 45.5 ± 3.3
c
Indirect 140 °C/4 s 93.6 ± 1.5c 70.0 ± 6.1
d
6.3.1.2 Caseins
Compared to the whey proteins, the caseins and casein micelles are very heat stable and the
micellar conformation is preserved even during severe heat treatments (Walstra, et al., 2006).
During severe heat treatments, polymerisation of caseins can occur, which could be caused by the
formation of lysinoalanine, isopeptide formation or the Maillard reaction (Zin El-Din & Aoki,
1993). The most important heat induced change of caseins during heat treatment is an increase in
the casein micelle size due to the association of denatured whey proteins (Anema & Li, 2003a;
Anema, 2007; Crudden, Oliveira, et al., 2005a). The increase in casein micelle size depends
largely on the degree of whey protein denaturation. Only small changes are observed when less
than 80 % of the whey proteins are denatured, while a fast and larger increase in micelle size is
observed at higher degrees of denaturation (Anema & Li, 2003b).
6.3.2 Heat inactivation of the plasmin system
Inactivation by heat of plasmin and plasminogen in milk has been closely linked to the
denaturation of β-lg and thiol-disulphide interchange reactions. Plasmin inactivation in the
absence of β-lg is decreased (Rollema & Poll, 1986) and a positive correlation between the
concentration of β-lg and the rate of plasmin activation has been found (Saint-Denis, et al.,
2001a). Proof of the thiol-disulphide reaction as the main driving force for plasmin inactivation
was provided by the increased inactivation of plasmin upon addition of cysteine (Durkee &
Hayes, 2008; Lu, R., et al., 2009; Metwalli, et al., 1998; Rollema & Poll, 1986) and decreased
plasmin inactivation upon blocking the free thiol group of β-lg by addition of KIO3 (Kelly &
Foley, 1997). The heat inactivation kinetics of plasmin and plasminogen in various dairy systems
has been investigated by several authors (Alichanidis, et al., 1986; Crudden, Oliveira, et al.,
46
2005a, 2005b; Metwalli, et al., 1998; Rollema & Poll, 1986; Saint-Denis, et al., 2001a). A
selection of kinetic parameters is summarized in an Arrhenius plot in Figure 20.
Figure 20: Arrhenius plot for the heat inactivation of plasmin in milk (red line) from Saint-Denis et al (2001) and plasmin+plasminogen in milk (green line) and a casein micelle solution (black line) from Rollema et al. (1986). The Arrhenius plot for heat denaturation of β-lg is indicated (dashed line) using the kinetic parameters of Dannenberg & Kessler (1988).
In milk, inactivation of plasmin and plasminogen shows a similar kinetic pattern to that of β-lg
denaturation, with a discontinuity at around 95 °C, indicating that the inactivation of plasmin and
plasminogen consists of two reactions with different activation energies as well. Older studies
report the discontinuity at 77 °C (Driessen & van der Waals, 1978; Metwalli, et al., 1998). The
difference in the kinetic reported by Rollema et al. (1986) and Saint-Denis et al. (2001b) above
90 °C could be attributed to the different assays used and the fact that the kinetics of Rollema et
al. (1986) are a combined kinetic for plasmin and plasminogen. Plasminogen exhibited a lower
heat stability compared to plasmin below 90 °C, but a higher stability above 90 °C is found by
Rollema et al. (1986), while Saint-Denis et al. (2001b) show comparable heat stabilities of
plasmin and plasminogen. Comparing the rate of β-lg denaturation with plasmin inactivation, it
becomes obvious that the rate for plasmin inactivation below 90 °C is much faster compared to
the denaturation rate of β-lg. Above 90 °C, the rates are very similar and, depending on the
applied inactivation kinetic, β-lg denaturation is actually faster than plasmin inactivation above
120 °C according to Saint-Denis et al. (2001b). As previously discussed, interaction of β-lg with
the casein micelle is less pronounced at higher temperatures. Since plasmin and plasminogen are
47
associated with the casein micelle, the same mechanism could apply for the inactivation of
plasmin and plasminogen. A reported comparison of plasmin inactivation and β-lg association
with the casein micelle suggested that the interaction of plasmin with β-lg and the β-lg
association with the casein micelle was faster than aggregation of β-lg in the temperature range
between 65-80 °C (Crudden, Oliveira, et al., 2005a). Direct UHT treatment with a pre-heat at
72 °C for 180 s and direct steam infusion at >150 °C for < 0.2 s resulted in an almost equal
degree of β-lg denaturation and plasmin activation (Paper II).
In comparison to the inactivation kinetic of plasmin in milk, the activation energy for the
inactivation of plasmin and plasminogen in a casein micelle solution is constant and the
inactivation rate is decreased by a factor of 5-10 in the casein micelle solution (Rollema & Poll,
1986). The same effect has been observed for the denaturation kinetic of α-la in absence of β-lg
(Tolkach, 2007; Wang, Q., et al., 2006). Based on the two step reaction kinetic consisting of an
unfolding and aggregation reaction, Tolkach (2007) concluded that the unfolding of α-la should
still result in a slight discontinuity of the activation energy in absence of β-g. While this is not the
case for isolated α-la, Metwalli et al. (1998) found a discontinuity in the inactivation kinetic of
isolated plasmin at 77 °C, indicating that the unfolding of plasmin has an effect on the
inactivation of plasmin. The difference to the results of Rollema et al. (1986) could be due to the
presence of caseins, which affect the inactivation of plasmin as well.
Figure 21 gives a good overview of the factors influencing plasmin inactivation in a milk system.
Plasmin inactivation in the presence of sodium caseinate is reduced compared to isolated plasmin.
Caseins are also able to protect plasmin against inactivation in the presence of β-lg, but not
against inactivation by cysteine (Metwalli, et al., 1998). The protective effect of caseins against
inactivation of plasmin is not fully understood yet. In absence of free thiol groups, caseins could
bind to the lysine binding sites of plasmin, which has been shown to enhance the heat resistance
of plasmin against inactivation by EACA (Grufferty & Fox, 1988a). Caseins have also been
shown to act as chaperones and could reduce the heat induced denaturation and inactivation or
assist in a refolding after heat treatment (He, et al., 2011; Morgan, et al., 2005; Treweek, et al.,
2011; Zhang, et al., 2005). This could especially be the case for the sodium caseinate used by
Metwalli et al. (1998). Metwalli et al. (1998) on the other hand showed that temperature-activity
of plasmin is not affected by caseins, suggesting that caseins do not protect plasmin from
unfolding. However, the unfolding and inactivation of plasmin in the absence of free thiol groups
does not necessarily include the unfolding of the light chain containing the active centre, but
could also involve aggregation or structural changes in the kringle structures.
48
Figure 21: Effect of two milk proteins on heat-induced inactivation of plasmin in a milk salt
solution. (A) Effect of sodium caseinate (+), β-lg (▴ ) and β-lg+caseinate (♦); (B) of sodium caseinate (+), cysteine (□), cysteine+caseinate (○), on inactivation of plasmin (control, ●) at 77 °C. From Metwalli et al. (1998).
In the presence of free thiol groups, a different protective effect of caseins seems to take place.
The fact that caseins are able to protect plasmin against inactivation by β-lg, but not by cysteine
implies a protection via a steric hindrance of the reaction between β-lg and plasmin by the
caseins. In this project, an experiment was planned to study the heat stability of plasmin
associated with caseins and plasmin released by NaCl and EACA. Using the protocol by Politis et
al. (1993) to dissociate plasmin, we could not detect any significant difference between the
inactivation rate of dissociated and associated plasmin at 80 °C in skim milk. Ultracentrifugation
of the milk, however, showed that approximately 50 % of plasmin was still attached to the
caseins. Due to lack of time, this could not be investigated further. Clarification of the protective
effect of caseins on plasmin inactivation could help to develop processes with improved plasmin
inactivation.
The fat content of milk influenced the heat inactivation of plasmin using lenient steam infusion
heat treatment (Dickow, 2011). A possible explanation for this could be the relatively high
number of free thiol groups on the MFGM, which could catalyse and enhance thiol-disulphide
interchange reactions during heat treatment (Lee & Sherbon, 2002). Plasmin inactivation in whey
was found to be very dependent on the type of whey. Plasmin inactivation in acid whey was
much slower compared to sweet whey, which could partially be attributed to the difference in pH
(Crudden, Oliveira, et al., 2005b). Interestingly, neither the inactivation kinetic of plasmin nor of
49
β-lg in acid whey showed the characteristic discontinuity found for milk (Crudden, Oliveira, et
al., 2005b).
Results on the heat inactivation kinetic of plasminogen activators are inconsistent. Overall, it is
known that plasminogen activators show slightly higher heat stability compared to plasmin (Lu,
D. D. & Nielsen, 1993b; Saint-Denis, et al., 2001a). While Saint-Denis et al. (2001b) find the
same kinetic for plasminogen activators compared to plasmin with a discontinuity at 90 °C,
whereas Lu & Nielsen (1993) report a single reaction with constant activation energy. While
Saint-Denis et al. (2001a) assay the plasminogen activators directly in milk, whereas Lu &
Nielsen (1993) use a milk model system containing buffer and casein. The absence of β-lg can
explain the difference in the inactivation kinetic. uPA and tPA contain 12 and 17 disulphide
bonds respectively, indicating that the inactivation mechanism is most likely similar to plasmin
and plasminogen. Heat inactivation of tPA and uPA shows that the latter is more heat stable
(Prado, et al., 2007). This could be due to the higher number of disulphide bonds in tPA
compared to uPA.
Similar to plasmin activity, the term ‘inactivation’ of plasmin system components is ambiguous.
Thiol-disulphide interchange reaction of β-lg with the kringle structure of tPA would result in a
largely decreased activity since tPA would no longer be able to bind to the caseins. The kringle
structure of uPA on the other hand is not important for the binding to caseins and a reaction with
β-lg would most likely not affect its activity. An assay using fibrin to enhance tPA activity
(Prado, et al., 2007) thus could lead to false assumptions regarding inactivation or reduced
activity of tPA. The same applies for the inactivation of plasmin. Disruption of the kringle
structures by thiol-disulphide interchange reactions could release plasmin and inhibit its action on
caseins, similar to the effect of EACA on plasmin activity towards fibrin. The activity in an assay
using a small synthetic substrate would however not be affected.
6.3.3 Effect of cysteine on plasmin and milk proteins during heat treatment
The addition of cysteine to milk has been proposed in several studies to improve the inactivation
of plasmin, however these studies do not specify the impact of cysteine addition on flavour and
milk proteins (Durkee & Hayes, 2008; Rollema & Poll, 1986). The effect of cysteine or other
reducing agents on milk proteins during heat treatment is largely unknown and has only been
studied in relation to acid gel formation after severe heating conditions (Goddard, 1996; Nguyen,
et al., 2012; Nguyen, et al., 2013). In order to evaluate the practical use of cysteine for improved
plasmin inactivation, the potential changes in milk proteins need to be considered. In an initial
study, low concentrations of cysteine up to 1.5 µM were added to pasteurized skim milk and heat
50
treated at 80 °C with holding times up to 60 s in a water bath. Plasmin inactivation increased with
increasing cysteine concentration as expected (Figure 22A).
Figure 22: Effect of addition of 0 (●), 0.5 (○), 1 (▼) and 1.5 (∆) µM cysteine on the inactivation of plasmin (A), monomeric κ-casein (B) and denaturation of β-lg (C) and α-la (D) during heat treatment at 80 °C. The sample needed approximately 10 s to heat up and the unheated sample is marked as -10 s.
A considerable inactivation of plasmin already occurred during heating up of the sample in the
first 10 s. In milk with 0.5 µM cysteine, a shift in reaction kinetic was observed after 30 s, which
indicated that the cysteine available for plasmin inactivation had reacted; thereafter inactivation
occurred at a similar rate as in the control sample without cysteine. The protein composition was
analysed as described in Paper II, with the exception that no reducing agent was added to the
urea and sodium citrate buffer. In samples containing cysteine, a peak appeared at the retention
time of unreduced κ-casein and was confirmed by MS-analysis as a monomeric κ-casein with a
mass shift of -2 Da compared to the reduced κ-casein, indicating an intramolecular disulphide
bond. The concentration hereof depended both on the holding time and on the cysteine
concentration (Figure 22B). This indicated a decrease of multimeric κ-casein by increased
formation of intramolecular disulphide bonds catalysed by cysteine as reported by Nguyen et al
(2012). In the sample with 1.5 µM cysteine, the concentration of monomeric κ-casein decreased
51
after 60 s holding time and a different peak appeared which was identified to be κ-casein with
two cysteine residues attached, suggesting a time and cysteine concentration dependent formation
of monomeric κ-casein and/or monomeric κ-casein with two cysteine residues.
Surprisingly, the degree of β-lg denaturation was only slightly affected by the presence of
cysteine (Figure 22C). Milk heat treated with higher cysteine concentrations showed slightly
increased β-lg denaturation, but no difference could be seen for 0.5 µM cysteine addition
compared with the control milk. In contrast to β-lg, the degree of α-la denaturation increased with
increasing cysteine addition (Figure 22D). The difference in the denaturation of β-lg and α-la
could be related to their structure and number of cysteine residues. The main mechanism for α-la
denaturation in milk is the thiol-disulphide interchange of α-la with the free thiol group of β-lg
and the presence of additional free thiol groups accelerates the denaturation (Tolkach, 2007). The
relatively small change in the denaturation of β-lg could be attributed to the free thiol group of β-
lg that might act as a catalyst to regain the native structure by releasing cysteine. Reaction of
cysteine with the free thiol group of β-lg resulted in denaturation of β-lg since β-lg with an
attached cysteine was present in total protein, but not in the pH 4.6 soluble material.
The differently treated milks were not evaluated sensorically, but all milk samples with added
cysteine had a distinct sulphuric smell. An immediate repetition of the trial was not possible since
the analysis of unreduced heat treated milk samples rendered the HPLC column unusable and no
appropriate column was available. The trial showed that the addition of cysteine significantly
affects milk proteins. In regards to a practical use of cysteine for the heat inactivation of plasmin
in milk products, these effects need to be further studied and characterized. While the
modifications and denaturation of whey proteins might not be a crucial factor for UHT milk, the
modifications of κ-casein could affect the stability of the casein micelle and result in
sedimentation or other quality defects.
52
6.3.4 Maillard reaction
The Maillard reaction is a chemical reaction of reducing sugars and amino groups. In milk, the
Maillard reaction occurs mainly during the heat treatment, but also during subsequent storage,
although at a much lower rate (see section 6.4.3). The main reducing sugar in milk is lactose and
it reacts mainly with lysine residues on proteins since the level of free amino acids in milk is
relatively low (Van Boekel, 1998). The Maillard reaction is sometimes divided into an early, an
advanced and a final stage.
Figure 23: Formation of the Amadori product lactulosyllysine by the reaction of a protein bound lysine with lactose. From Siciliano et al. (2013).
The early stage of the Maillard reaction, depicted in Figure 23, is the condensation of the
reducing sugar with the amino group and the formation of a Schiff’s base, which following
Amadori rearrangement gives the Amadori product lactulosyllysine (Van Boekel, 1998).
In the advanced stage of the Maillard reaction, the Amadori product breaks down into numerous
products. The final stage consists of the condensation of amino and sugar compounds to
polymerized proteins and brown pigments.
The Maillard reaction can significantly affect the quality of milk and milk products. Blockade of
the lysine residues reduces the nutritional value and digestibility (Dalsgaard, et al., 2007; Van
Boekel, 1998). Various flavour compounds and compounds with either beneficial (antioxidative,
antimicrobial) or harmful (mutagenic, allergenic) properties can be formed (Siciliano, et al.,
2013; Van Boekel, 1998). Colour changes occur when brown pigments are formed. Additionally,
the functionality of proteins can be changed by lactosylation, such as solubility and thermal
stability (Wang & Ismail, 2012). The heat load applied during UHT processing is usually not
sufficient to produce the final Maillard reaction products and colour changes. The main Maillard
reaction product found in UHT milk is the Amadori product. Some advanced Maillard reaction
products, such as 5-hydroxyl-2-methylfurfural (HMF), are present in UHT milk, but only in a
limited amount (Birlouez-Aragon, et al., 1998; Claeys, et al., 2003, 2004).
53
Direct analysis of the Amadori product is difficult, as it can only be measured after enzymatic
breakdown of the lactosylated proteins. The most common way to measure protein lactosylation
is to measure furosine, a product that is formed by acid hydrolysis of the Amadori product
(Erbersdobler & Somoza, 2007; Van Boekel, 1998). A different approach is the boiling of the
lactosylated proteins in oxalic acid to induce the formation of HMF. Both methods have the
disadvantage that only a part of the Amadori product is actually converted to furosine and HMF,
and HMF itself is already present in milk. In addition, the sample preparation is a very time
intensive process.
The formation of lactulosyllysine results in a mass change of the unmodified protein of +324 Da
and can be measured by mass spectrometry (Figure 23). Several studies investigated the
lactosylation of whey proteins and caseins (Czerwenka, et al., 2006; Meltretter, et al., 2009;
Scaloni, et al., 2002; Siciliano, et al., 2013). While a qualitative assessment of protein
lactosylation can easily be achieved, quantification of the extent of lactosylation using MS can be
challenging. The quantification of protein lactosylation will be discussed in detail in section
6.4.3.
6.3.5 Lactulose
Besides whey protein denaturation, furosine and HMF, lactulose is often used as marker for the
heat treatment of UHT milk (Datta, et al., 2002). Lactulose is an isomeric form of lactose and is
formed during heat treatment. The reaction is catalysed by either amino groups of the milk
proteins or by inorganic salts (Burton, 1994b; Datta, et al., 2002). Lactulose is a stable product
and not present in raw milk. However, the formation of lactulose is very pH dependent (Burton,
1994b).
54
6.4 Shelf life development of UHT milk
6.4.1 Age gelation of UHT milk
The term ‘age gelation’ has been used in literature to describe the phenomena of physical
instability of sterilized milk. Age gelation observed by different researchers differs greatly in gels
appearance and the cause and/or mechanism by which they are formed (Nieuwenhuijse & van
Boekel, 2003). The mechanisms of age gelation have been divided into a proteinase-induced,
non-enzymatic-induced and a two stage process consisting of a combination of proteolysis
followed by physical chemical changes (Aroonkamonsri, 1996; Harwalkar, 1992; Manji &
Kakuda, 1988). An overview of causes and mechanisms of age gelation by Nieuwenhuijse & van
Boekel (2003) is shown in Figure 24.
Figure 24: Simplified scheme of various pathways of protein destabilisation in milk and milk products. From Nieuwenhuijse & van Boekel, 2003).
Non-enzymatic age gelation in normal UHT milk is extremely slow, while fast in concentrated
milks (Manji & Kakuda, 1986; Nieuwenhuijse & van Boekel, 2003). The proposed mechanism
for age gelation at medium and high storage temperatures in concentrated UHT milks is the
55
reaction between β-lg and κ-casein during the heat treatment. This complex can dissociate from
the casein micelle during subsequent storage and form a gel network (McMahon, 1996). In age
gelation of concentrated UHT milk, protein dissociation and casein micelle crosslinking are
regarded as factors causing age gelation. The hypothesis that proteolysis merely facilitates the
release of β-lg-κ-casein complexes from the casein micelle is untenable (Datta & Deeth, 2001,
2003; Malmgren, 2007; McMahon, 1996). Various studies, including Paper II, show that
protease induced age gelation is actually impaired by increasing heat treatment. Furthermore,
Paper II showed that age gelation is possible with almost no β-lg associated to κ-casein, findings
in accordance to (Kelly & Foley, 1997; Manji & Kakuda, 1988).
Protease induced age gelation can be caused both by plasmin and heat stable proteases originating
from psychotrophic bacteria. In contrast to plasmin, bacterial proteases are able to hydrolyse κ-
casein and can, in a similar fashion as chymosin, result in a rennet-curd like gel (Nieuwenhuijse
& van Boekel, 2003; Snoeren, et al., 1979). Recent studies showed that bacterial proteases,
besides hydrolysing κ-casein, also are able to destabilize casein micelles by proteolysis in general
(Gaucher, et al., 2009; Gaucher, et al., 2011). Bacterial proteases show a more unspecific
proteolysis pattern, which usually results in small peptides and can be distinguished from plasmin
peptides by different RP-HPLC preparation methods. While both peptides formed by bacterial
proteases and plasmin are soluble at pH 4.6, the larger plasmin derived peptides are not soluble in
TCA (Datta & Deeth, 2003).
The role of plasmin in age gelation has been subject of intensive research, but it has not been
unequivocally proven that plasmin can cause age gelation (O’Mahony, et al., 2013). Although
plasmin has been shown to be involved in age gelation, the conditions under which gelation
occurs vary considerable and so far there is no direct correlation between the onset time of
gelation and level of proteolysis (Manji & Kakuda, 1988). As the study described in Paper II
shows, age gelation can occur in UHT milk with residual plasmin activity. No age gelation takes
place when heat treatment conditions result in complete inactivation of plasmin and plasminogen,
or when plasmin activity is inhibited (de Koning, et al., 1985; Kohlmann, et al., 1988; Manji &
Kakuda, 1986). Age gelation in relation to plasmin therefore occurs more frequent when using
direct UHT systems compared to indirect systems (Datta, et al., 2002; Manji & Kakuda, 1986). A
first crucial factor for age gelation caused by plasmin is the rate of proteolysis. A high rate of
proteolysis, either by high residual plasmin activity or addition of plasmin, plasminogen or
somatic cell extracts results in a solution of polypeptides, but not in gelation. Addition of
different concentrations of plasmin shows that high levels of plasmin activity result in 2-phase
separation with clarification of milk and creaming (Crudden, Afoufa-Bastien, et al., 2005;
Kohlmann, et al., 1991). Addition of low concentration of plasmin or plasminogen on the other
56
hand results in gel formation (Kohlmann, et al., 1991). Similar effects are shown, when somatic
cell extracts are added to milk (Kelly & Foley, 1997). A second impact factor is the storage
temperature of UHT milk. Gelation is slow at low storage temperatures, most rapid at 20-25 °C,
and does not or very late occur at storage at > 30 °C (Harwalkar, 1992; Malmgren, 2007; Manji
& Kakuda, 1986; Nieuwenhuijse & van Boekel, 2003). Since proteolysis was on-going in these
UHT milks, it is assumable that proteolysis by plasmin was too fast to allow gel formation at
storage temperatures > 30 °C (Nieuwenhuijse & van Boekel, 2003). Manji & Kakuda (1988) tried
to separate the effect of proteolysis from the effect of storage temperature and time by pre-
hydrolysing milk with plasmin to different degrees before storage. They found that gelation
occurred faster at higher storage temperatures, but did not find a correlation between the time of
gelation and the degree of hydrolysis.
Plasmin induced age gelation is therefore most likely a two-step process, consisting of the
proteolysis itself and physical-chemical changes in the casein structure. In the UHT trial
conducted for Paper II and III, two UHT milks with plasmin activity were produced: The UHT
milk described in Paper II with a pre-heat treatment of 72 °C for 180 s and a UHT milk pre-heat
treated at 72 °C for 5 s. While two replicates of the latter UHT milk were identical and showed
comparable plasmin activities and proteolysis compared to the milk used for Paper II and III,
one replicate showed decreased plasmin activity and reduced proteolysis of caseins and peptide
formation (Figure 25).
57
Figure 25: Hydrolysis of αS1-casein (A), development of β-casein f(1-28) (B), plasmin activity (C) and viscosity development (D) in three replicate UHT milks (●,○,□) heat treated at >150 °C for > 0.2 s with a pre-heat treatment of 72 °C for 5 s during storage.
The time point of gelation, analysed as viscosity increase, in this replicate was also delayed by 2-
3 weeks compared to the other two replicates (Figure 25D) and occurred when the same level of
αS1-casein had been hydrolysed (Figure 25A). This indicates that at least in a narrow range, the
extent of proteolysis does have an effect on the time point of gelation.
In conclusion, the plasmin induced age gelation depends mainly on the following critical
parameters at a given storage temperature:
Minimum degree of proteolysis to sufficiently destabilize the casein micelle.
Minimum storage time for changes in casein micelle structure to form a gel which
mainly depends on the storage temperature.
Maximum degree of proteolysis so that the peptides are no longer able to form a gel
matrix any longer.
58
Although this is a very simplified model, these three factors could explain the different findings
in relation to storage time and temperature and degree of proteolysis upon gelation. A schematic
illustration of the model is shown in Figure 26:
Figure 26: Schematic illustration of the critical parameters for plasmin induced age gelation as indicated by dashed lines: Maximum degree of proteolysis (1), minimum degree of proteolysis (2) and minimum storage time (3).
Other factors could influence these parameters as well. Proteolysis correlated well with a
decrease in pH, which could affect the calcium equilibrium in milk and the stability of the casein
micelle. Whether such changes are sufficient to affect casein micelle stability is unknown and the
effect of calcium on age gelation by plasmin is limited (Nieuwenhuijse & van Boekel, 2003).
Another factor could be the activation of plasminogen and the previously discussed change in the
rate of proteolysis, which makes the comparison of UHT milk systems and model systems with
added plasmin difficult. Destabilisation of the casein micelle could also change the proteolytic
pattern of plasmin, as indicated in Paper III, and proteolysis and destabilisation could have large
influences on each other.
In contrast to age gelation by bacterial proteases, the mechanism of casein micelle destabilisation
and gel formation as a consequence of plasmin activity is largely unknown. Electron micrographs
of plasmin induced gels showed partially disintegrated casein micelles and the gel was made of
threads and knots of protein (Aroonkamonsri, 1996; de Koning, et al., 1985).The presence of
homogenized fat globules with a size distribution similar to casein micelles interfered with the
particle size measurement of casein micelles in the study presented in Paper II. We could
therefore not investigate changes in the particle size distribution of casein micelles, which could
indicate disintegration caused by proteolysis. An attempt was made to interpret the observed
decrease in Lab-colour values with changes in casein micelle structure. Dilution of UHT milk
59
without proteolysis with water resulted in a decrease of the b-value, i.e. less yellow, while the L-
and a-values remained constant. Dilution with sodium citrate and dissociation of the casein
micelles resulted in a decrease of L-, a-, and b-values similar to the UHT milk with plasmin
activity, which could indicate a disruption and dissociation of casein micelles due to proteolysis.
The initial gel is usually formed at the bottom of the container and has been described as fragile
and custardy (Aroonkamonsri, 1996; Datta & Deeth, 2003; Kelly & Foley, 1997). The gel found
in the UHT milk described in Paper II showed similar features as can be seen in Figure 27.
Figure 27: Effect of Storage time on gel formation in the bottom of the bottles in 3 replicates of UHT milk in Paper II. Ages of milk: 13, 12 and 11 weeks (from left to right). Notice the increase in gel volume over time.
The gel at the bottom of the bottle increased in volume during the storage time and after
approximately 20 weeks, the entire bottle was gelled. Although the UHT milk was not analysed
further, some bottles were kept for visual inspection and after 25 weeks, a clear phase separation
between the gel and the serum phase was observed. According to Nieuwenhuijse & van Boekel
(2003), gelation is dependent on the dissociation and aggregation of peptides since non-casein
nitrogen (NCN) increases upon proteolysis by plasmin. Analysis of pH 4.6 soluble peptides
showed that main polypeptides found in the serum were proteose peptones. The only large
polypeptide deriving from αS1-casein was f(125-199) and it was associated with caseins.
Comparison of protein and peptide compositions of gel and liquid phases showed a similar
composition, but the gel contained lower concentrations of κ-casein. The main bond type to
stabilize a plasmin induced gel seems to be hydrophobic interaction (Aroonkamonsri, 1996;
Nieuwenhuijse & van Boekel, 2003).
60
In Paper II, we compared the gelation mechanism of plasmin induced gelation with acid gelation
of milk, which is caused by reduction of charge repulsion and solubilisation of colloidal calcium
phosphate (Lucey, et al., 2001; Lucey & Singh, 2003). The plasmin induced gel found in the
UHT milk exhibited a similar microstructure as found in acid gels (Figure 28). The proteolytic
pattern of plasmin on the other hand showed that plasmin hydrolysed caseins preferably in
hydrophilic domains and at the border of hydrophilic-hydrophobic or hydrophilic-phosphoserine
regions (Paper III).
Figure 28: CLSM micrographs of gel particles extracted from gelled UHT milk after 13 weeks of storage at different magnifications. Protein is stained green, fat is stained red. Fat globule clusters and their incorporation in the gel network can be seen. From Paper II.
This indicates that plasmin could destabilize the casein micelle by hydrolysing around regions,
which stabilize the casein micelle by calcium phosphate and hydrophobic interactions. It could be
hypothesized that this proteolysis pattern could result in a disruption of the casein micelle, when
enough stabilizing regions are hydrolysed. Loss of the casein micelle structure could
subsequently result in a rearrangement of the polypeptides to a gel like network.
The exact mechanism of plasmin induced age gelation still remains unknown. The UHT trial
conducted in this project aimed to investigate the effect of plasmin on UHT milk in general and it
was not known beforehand, whether gelation actually would occur. In order to investigate the
61
relation between proteolysis and gelation, UHT milks with a broad range of residual plasmin
activities should be produced. The level of residual plasmin activity could be easier controlled by
choosing one pre-heat temperature and changing the holding time to create appropriate residual
plasmin activities. To study gelation mechanisms, especially destabilisation of casein micelles,
the use of skim milk as feed would allow to observe changes in particle size. The release of
caseins or peptides in relation to proteolysis could be achieved by separating the serum phase
from casein micelles by ultracentrifugation and analysis of both fractions for peptides and intact
caseins. A deeper investigation of the gel by either addition of citrate or cooling or transferring
the gel into a hydrophobic medium could allow insight to, whether calcium or hydrophobic bonds
stabilize the gel.
6.4.2 Flavour development
6.4.2.1 Chemical changes affecting flavour development
Freshly prepared UHT milk has a distinctive cooked, sulphurous flavour, which disappears
during storage. At the same time, an aged and stale flavour develops (Figure X). In addition,
flavour attributes such as ‘heated’ and ‘oxidized’ develop. The flavour of UHT milk is commonly
perceived as poor and is a problem for the dairy industry since the customer acceptance for UHT
milk is low (Colahan-Sederstrom & Peterson, 2004; Perkins, M. L. & Deeth, 2001).
Figure 29: Diagrammatic representation of flavour changes in UHT milk during storage. Adapted from Burton (1994).
The initial cooked flavour arises from volatile sulphur components, which are formed during the
UHT treatment. The major source of these sulphur components are free thiol groups, particularly
of β-lg. The MFGM is also rich in free thiol groups and the concentration of volatile sulphur
62
components increases with increasing fat content (Al-Attabi, et al., 2009; Vazquez-Landaverde,
et al., 2006). In addition, several volatiles can be formed during the Maillard reaction with
methionine and cysteine (Al-Attabi, et al., 2009). The main components responsible for the
cooked flavour are methanethiol, dimethylsulphide and dimethyldisulphide. Hydrogen sulphide
itself has a relatively high odour activity value, but can react further to other sulphur volatiles
(Vazquez-Landaverde, et al., 2006; Zabbia, et al., 2011). Disappearance of cooked flavour is
dependent on the concentration of oxygen in UHT milk. In the presence of oxygen, volatile
sulphur components are rapidly oxidized and the cooked flavour decreases (Al-Attabi, et al.,
2009; Burton, 1994a). Directly processed UHT milk usually has a lower initial cooked flavour
compared to indirectly processed UHT milk. This is due to the lower overall heat load in directly
processed UHT milk and some of the volatile components can be removed during the vacuum
cooling step (Datta, et al., 2002). On the other hand, most of the oxygen is removed during
vacuum cooling as well and the cooked flavour disappears slower compared to indirectly
processed UTH milk (Datta, et al., 2002; Valero, et al., 2001).
High levels of oxygen accelerate the appearance of stale and oxidized flavour. Stale and oxidized
flavours have been linked to the presence of lipid oxidation products, such as methyl ketones and
aldehydes (Calvo & de la Hoz, 1992; Perkins, Melinda L., et al., 2005; Valero, et al., 2001;
Vazquez-Landaverde, et al., 2005). Aldehydes are regarded to have a higher impact on flavour
than methyl ketones due to their lower sensory threshold (Datta, et al., 2002). Oxidation of
proteins can also lead to an unpleasant oxidized or ‘activated flavour’ (Burton, 1994a). Methyl
ketones also form in the Maillard reaction and contribute to the heated flavour of UHT milk.
Other Maillard reaction products contributing to the flavour are lactones, diacetyl, maltol, vanillin
and benzaldehyde (Calvo & de la Hoz, 1992; Contarini, et al., 1997; Jansson, Jensen, et al.,
2014).
6.4.2.2 Effect of enzymes on flavour
Flavour development of UHT milk also depends on raw milk quality. Psychotrophic bacteria are
able to produce heat resistant lipases, whose activity can lead to a rancid flavour (Burton, 1994a).
Proteolysis can indirectly affect the flavour of UHT milk. Increased levels of free amino acids
accelerate the Maillard reaction and lead to a higher concentration of Strecker degradation
products (Jansson, Clausen, et al., 2014).
Several studies reported the occurrence of bitterness before gelation of milk in stored UHT milks
(Burton, 1994a; McKellar, et al., 1984; Nieuwenhuijse & van Boekel, 2003). While many studies
63
focussed on age gelation of milk, studies on bitterness in milk are scarcer. Since bitterness occurs
before gelation, it is the shelf life limiting factor and of higher importance for the dairy industry.
Bacterial proteases are found to cause bitterness at very low levels of proteolysis (McKellar,
1981). These proteases cleave relatively unspecific and are able to hydrolyse hydrophobic
domains of the caseins, which are the largest source of bitter peptides (Gaucher, et al., 2011;
Lemieux & Simard, 1992). The appearance of bitterness thus has mostly been connected to the
presence of bacterial proteases.
The appearance of bitterness in the UHT milk described in Paper II was linked to plasmin since
the milk was as fresh as possible, of excellent microbial quality and proteolysis of bacterial
proteases was not detected during storage (Paper III). Huijs et al. (2004) report plasmin as the
cause for bitterness in directly processed UHT milk with ultra-short holding time, similar to the
process used in Paper II. Bitterness is also observed in directly processed UHT milk with high
somatic cell count and plasmin activity (Santos, et al., 2003; Topçu, et al., 2006). The milk also
contained a high level of psychotrophic bacteria so the exact cause of bitterness cannot be
differentiated.
The literature only describes one sensory evaluation of peptides produced by plasmin and it is
done on isolated β-casein (Harwalkar, et al., 1993). Proteolysis of β-casein caused an astringent
flavor and was linked to the formation of γ-caseins. Peptides from tryptic digests of β-casein were
described as ’astringent, tickling, burning and bitter’, verifying that proteolysis of β-casein by
plasmin could lead to bitter peptides (Bumberger & Belitz, 1993).
A common way to estimate the bitterness of a peptide is the Q-rule. Bitterness has for long been
associated with the hydrophobicity of a peptide. The Q-value is an average value of the
hydrophobicity of the amino acid side chains divided by the number of amino acids (Ney, 1971).
Peptides with a Q-value < 1300 cal res-1
are not bitter while peptides with Q > 1400 cal res-1
are
generally considered as bitter. No prediction of bitterness is possible for Q-values between 1300-
1400 cal res-1
(Lemieux & Simard, 1992; Ney, 1971). In Paper III, we identified 23 potentially
bitter peptides based on the Q-rule. With the exception of β-casein f(49-97), the potential bitter
peptides originating from β-casein found in the UHT milk were not described as bitter in tryptic
digests (Bumberger & Belitz, 1993). Interestingly, the most bitter peptide found in tryptic digests
of β-casein, β-casein f(203-209), was not identified in UHT milk (Bumberger & Belitz, 1993).
The C-terminal region of β-casein is very hydrophobic and it was suggested that it is inaccessible
for plasmin in a milk system. Most of the peptides, also previously proposed bitter peptides,
derived from hydrophobic regions of αS1- and αS2-casein. (Habibi‐Najafi, et al., 1996; Le Bars &
Gripon, 1989; Maehashi & Huang, 2009).
64
Although the Q-rule allows for an initial assessment of potential bitter peptides, it was not
possible to ascribe the observed bitterness in the UHT milk to certain peptides. An exception to
the Q-rule is that it is only valid up to a molecular weight of 6000 Da (Lemieux & Simard, 1992;
Ney, 1971). In order to identify the sensory properties of these potential bitter peptides, they
would need to be either extracted from milk or synthesized and evaluated sensorically. This
would have to be done in a milk system to include matrix effects, which could partially mask
bitter taste (Toelstede & Hofmann, 2008). Determination of the formation rate and quantification
of confirmed bitter peptides could allow a prediction of bitterness and consequently the shelf life
of UHT milk.
6.4.3 Lactosylation of milk proteins during storage of UHT milk
As indicated in section 6.3.4, the Maillard reaction takes place during storage of UHT milk at
ambient temperatures and results in an increase in protein lactosylation during storage. The rate
of protein lactosylation depends on the storage temperature with increased rates of protein
lactosylation at higher storage temperatures (Bosch, et al., 2008; Corzo, et al., 1994; Nangpal &
Reuter, 1990). Nangpal & Reuter (1990) reported a linear increase in furosine concentration in
UHT milk during storage at 20 and 30 °C, with an increase in furosine of 1 mg L-1
per week,
similar to the rate of furosine increase reported for the UHT milks in Paper IV (Figure 30). The
increase in furosine over time was found to be independent of the initial furosine concentration
(Paper IV). At higher storage temperatures, the increase in furosine is not linear any more at
prolonged storage times as lactulosyllysine reacts further and intermediate and late Maillard
reaction products are formed (Le, et al., 2011; Nangpal & Reuter, 1990).
Figure 30: Furosine development in directly heated UHT milk (> 150 °C for < 0.2 s) pre-heated at 95 °C for 5 (●) or 180 s (○) and in indirect UHT milk (140 °C for 4 s, □) during storage at 20 °C. Error bars indicate standard deviation, n = 3; from Paper IV.
65
Assessment of protein lactosylation by measurement of furosine includes an 18 h acid boiling
step to form furosine followed by analysis of furosine by HPLC, making this analysis expensive
and time intensive. Lactosylation can also be observed by LC-MS using the protein composition
method described in Paper II & IV. This method requires a one hour sample preparation step
and a HPLC run time of 20 min per sample and can give a more detailed view on the Maillard
reaction since the lactosylation of different proteins can be observed.
Figure 31: Summed mass spectra (retention time 11.0-13.2 min) of unmodified and lactosylated αS1-casein 8 P in indirect UHT milk (140 °C for 4 s) after 6 months of storage at 20 °C. Inset is showing a zoom of the most abundant ions. The m/z shifts due addition of lactose are indicated. From Paper IV.
Lactosylation renders a protein more hydrophilic and lactosylated proteins elute earlier in reverse
phase HPLC than non lactosylated proteins (Czerwenka, et al., 2006; Losito, et al., 2007; Losito,
et al., 2010). This results in a broadening of peaks in the UV chromatogram of proteins (Paper II
& IV). A chromatographic separation of lactosylated proteins, which would allow for
quantification of lactosylation using the UV signal, is hard to achieve since the change in
hydrophobicity upon lactosylation is little. Analysis of summed mass spectra of a peak containing
non lactosylated and lactosylated proteins on the other hand allows a relative comparison of the
MS intensities of the different lactosylated proteins. The comparison can either be performed on
specific ions (Figure 31) or on deconvoluted spectra (Figure 32)
66
Figure 32: Deconvoluted mass spectrum of αS1-casein 8 P in indirect UHT milk (140 °C for 4 s) after 0 (A) and 6 (B) months of storage at 20 °C. Mass shifts due to addition of lactose are indicated. From Paper IV.
Exploration of milk protein lactosylation has mainly been focused on whey proteins, especially
pH 4.6 soluble whey proteins (Siciliano, et al., 2013). This limits the characterisation of protein
lactosylation to milk products in which a sufficient amount of pH 4.6 soluble whey proteins are
present (Meltretter, et al., 2009). In products where protein lactosylation actually could affect the
overall product quality, the heat treatment is so severe that most or all of the whey proteins are
denatured, such as the indirectly processed UHT milk in Paper IV or milk powders.
Furthermore, denaturation and unfolding of whey proteins could affect the kinetic of lactosylation
due to increased susceptibility of lysine residues (Losito, et al., 2010). Still for a limited range of
heat treatments a quantitative relationship between lactosylation of whey proteins and heat
markers could be established. The used heat markers were whey protein denaturation as well as
the fluorescence of advanced Maillard products and soluble tryptophan (FAST) index (Losito, et
al., 2010; Meltretter, et al., 2009). While these markers reflect the severity of the heat treatment,
they do not necessarily correlate with the extent of the Maillard reaction. The only direct
correlation of milk protein lactosylation measured by MS and furosine concentration was
established for the lactosylation of β-casein in processed cheese (Steffan, et al., 2006). Other
studies on lactosylation of caseins measured by MS are only qualitative (Johnson, et al., 2011;
Scaloni, et al., 2002).
Quantitative analysis of milk protein lactosylation can be challenging since reports on response
factors of differently lactosylated protein species differ and quantitative comparison with furosine
is lacking in most studies. For proteins in their native state, such as whey proteins and lysozyme,
the response factor was found to be not affected by lactosylation of proteins when analysed with
67
both electrospray ionisation MS (ESI-MS) and matrix assisted laser desorption (MALDI) MS
(Czerwenka, et al., 2006; Meltretter, et al., 2009; Yeboah & Yaylayan, 2001). For reduced
caseins without any notable tertiary structure however, a lower response factor for lactosylated
proteins was found compared to non lactosylated caseins (Scaloni, et al., 2002). In Paper IV,
both lactosylated species of caseins and whey proteins exhibited a reduced response factor in MS
intensity, although the UV chromatogram showed no loss of protein and was constant throughout
the storage period. Although the response factors were not identical for different lactosylated
protein species in Paper IV, a linear regression of the relative area distribution of the proteins
was possible. The rate of lactosylation measured as decrease in unlactosylated protein correlated
with the number of lysine residues in protein for both caseins and whey proteins. The decrease of
unlactosylated proteins correlated well with the increase in furosine during storage, allowing a
quantitative estimation of protein lactosylation by LC-MS measurements in the covered range of
furosine.
The differences in response factors could be attributed to the structure of proteins. The globular
structure of whey proteins results in an overall decreased ionisation. A comparison of mass
spectra of pH 4.6 soluble whey proteins and reduced whey proteins in low pasteurized milk
samples showed large differences. The most abundant charge state for reduced β-lg and α-la were
17 and 13 compared to 11 and 6 for pH 4.6 soluble whey proteins. With increasing charge state, a
potential reduction in the ionisation efficiency of a lactosylated lysine residue would become
more pronounced compared to lower charge states, where ionisable residues are present in
excess. Analysis of the response factors of lactosylated pH 4.6 soluble whey proteins and
comparison to the response factors after reduction of the same samples could give an insight in
the underlying mechanism.
A different approach to determine the extent of lactosylation milk is the analysis of lactosylated
peptides present in milk (Pinto, et al., 2012). Plasmin derived peptides and especially β-casein
f(1-28) were found to be well suited to detect lactosylation in milk as they are already present in
raw milk. A high concentration of lactosylated β-casein f(1-28) was detected in the peptidomic
study of UHT milk with plasmin activity in Paper III, but the concentration of β-casein f(1-28)
was not high enough in the UHT milks without plasmin activity in Paper IV to obtain
reproducible results.
68
7 Conclusions
The studies presented in this thesis have provided insight into the measurement of plasmin
activity and the effect of plasmin activity on the shelf life development of UHT milk.
The tested conditions for measuring plasmin activity in an assay showed that the measured
plasmin activity is not only dependent on sample composition and treatment, but also on sample
preparation and buffer systems used. The term activity has been used in literature to describe
plasmin concentration, real activity and apparent activity including interfering components, but
should actually be referred to as the rate of hydrolysis of a synthetic substrate under given
conditions. There is no right or wrong way to measure activity, but one should be aware of all
factors that influence the activity in an assay in order to take decisions on assay conditions to be
used and to draw conclusions from activity measurements on changes in activity or the
characteristics of plasmin in milk. Considering these factors allows the design of assays that
measure an activity, which in the best way reflects the research aim.
Although it is more time and work intensive than an activity assay, following proteolysis by
plasmin gives a direct insight to the effect of plasmin activity in UHT milk. Identification of
peptides allowed identification of several marker peptides for plasmin activity, which were not
further hydrolysed during the presently applied storage period. Furthermore, differences in
affinity of plasmin for specific cleavage sites lead to a time dependent appearance of peptides,
which can potentially be used to characterize different stages of proteolysis. It is possible to
model the proteolysis of β-casein on a protein and peptide level, and of αS2-casein on a peptide
level with the analytical tools available in this project. Unfortunately, we could only model the
kinetic at one plasmin concentration and were not able to validate the model.
In terms of quality defects, plasmin activity could be linked to the occurrence of bitterness and
age gelation in UHT milk in this study. The identification of (potential) bitter peptides provides a
starting point for further studies on the sensory threshold of bitter peptides, which would allow
the establishment of critical peptide concentrations that can limit the shelf life of UHT milk.
Proteolysis by plasmin can cause age gelation of UHT milk, but the underlying mechanisms are
still not fully understood. The gelation mechanism is most likely a two-step process and the
proteolysis pattern leading to gelation can provide a hypothetical mechanism of destabilisation of
the casein micelle by proteolysis.
The quality defects in the UHT milk investigated in this study occurred only after substantial
proteolysis with 60-70 % of αS1- and β-casein being hydrolysed for the milk to taste bitter and
more than 90 % to cause gelation. While the extent of proteolysis necessary for gelation and
bitterness could not be answered for different residual plasmin levels, this study gave an insight
69
in the effect of plasmin activity in milk and provided a setup to explore this further. The results
show that low levels of plasmin activity in UTH milk can be acceptable, when the quality defects
are matched with the shelf life of the UHT milk.
70
8 Perspectives
The research effort on plasmin and the plasmin system in milk is considerable, and with every
published study, some questions are answered, but new and often more profound questions arise.
The same applies for this study.
The presented results give an insight into the effect of proteolysis on UHT milk, but cannot
answer the mechanism of age gelation or establish a direct connection between plasmin activity,
proteolysis and quality defects. In retro perspective, the UHT trial conducted in this study could
have been planned differently. Applying the same preheat temperature and only varying the
holding time would have allowed a more specific control over residual plasmin activities in the
UHT milk and maybe could have been predicted using kinetic data in literature. Sensory
evaluation of the UHT milk by a trained sensory panel would have given a more detailed view on
off flavour and bitterness caused by plasmin. Most potential bitter peptides were present in low
concentrations and could not be quantified using the UV signal. Quantification of these peptides
can be improved by optimizing the chromatographic separation or adapting the sample
preparation for smaller hydrophilic peptides. An alternative is the use of an internal standard for
the LC-MS analysis, which then allows quantification using the MS signal over a certain range.
In order to study the gelation phenomenon, a skim milk system and dynamic light scattering
instead of static light scattering should be used in order to investigate changes in the casein
micelle structure. Furthermore, release of peptides and caseins from the casein fraction could be
used as indicators for casein micelle destabilisation. A repetition of the UHT storage experiment
in this study under the conditions mentioned above could enable the correlation of plasmin
activity, proteolysis and quality defects and allow the dairy industry to control the effects of
plasmin activity better.
As indicated in Paper I, the dissociation of plasmin and caseins affect the interaction of plasmin
with inhibitory components. A dissociation of plasmin and caseins during processing, for
example by pressure, could result in an increased inhibition and/or inactivation of plasmin by
whey proteins and inhibitors. Although we could not see differences in the heat inactivation of
plasmin in the presence of NaCl and EACA, it could be hypothesized that the dissociation of
plasmin and caseins could negate the protective effect of caseins towards the heat inactivation of
plasmin.
Beside these follow up studies to this project, several more basic aspects of plasmin still need
more detailed attention. The concentration of plasminogen activators and the higher heat stability
of plasmin compared to plasminogen make them an important factor to limit proteolysis in milk
and dairy products. The influence of storage conditions of raw or pasteurized milk on the
71
activation of plasminogen before further processing has only been partially studied and could be a
starting point to reduce plasminogen activation. During the separation of milk into skim milk and
cream, somatic cells and other large particles are separated as well. Most of the uPA is initially
bound to somatic cells and the effect of the separation on the stability of somatic cells and release
of uPA is unknown. As uPA is considered the major plasminogen activator and has a higher heat
stability compared to tPA, a reduction of uPA concentration in milk would reduce proteolysis in
milk and milk products. Pre-processing of UHT milk yields several possibilities, which could
result in a minimisation of the plasmin system load in the final product, which could positively
affect product quality.
72
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Paper I
Rauh, V. M., Bakman, M., Ipsen, R., Paulsson, M., Kelly, A. L., Larsen, L. B., & Hammershøj,
M. (2014)
The determination of plasmin and plasminogen-derived activity in turbid samples from
various dairy products using an optimised spectrophotometric method
International Dairy Journal, 38, 74-80
The determination of plasmin and plasminogen-derived activity inturbid samples from various dairy products using an optimisedspectrophotometric method
Valentin M. Rauh a,b,*, Mette Bakman a, Richard Ipsen c, Marie Paulsson d, Alan L. Kelly e,Lotte B. Larsen b, Marianne Hammershøj b
aArla Foods Strategic Innovation Centre, Rørdrumvej 2, DK-8220 Brabrand, DenmarkbAarhus University, Dept. of Food Science, Faculty of Science and Technology, Blichers Allé 20, DK-8830 Tjele, DenmarkcUniversity of Copenhagen, Department of Food Science, Rolighedsvej 30, DK-1958 Frederiksberg C, DenmarkdDepartment of Food Technology, Engineering and Nutrition, Lund University, SE-221 00 Lund, Swedene School of Food and Nutritional Sciences, University College Cork, Cork, Ireland
a r t i c l e i n f o
Article history:Received 13 June 2013Received in revised form24 March 2014Accepted 26 March 2014Available online 26 April 2014
a b s t r a c t
A spectrophotometric assay for plasmin and plasminogen-derived activity in dairy products was opti-mised and extended to determine plasmin and plasminogen-derived activity in turbid samples of dairyproducts. The method was validated by assessing reproducibility, repeatability, level of detection andrecovery of plasmin activity in different sample matrices. Plasmin activity in raw milk was not affected byskimming, but decreased by 30% in pasteurised and homogenised whole milk, leading to an underes-timation of plasmin activity. The effects of dissociation of plasmin and caseins by ε-aminocaproic acid(EACA) plus NaCl on the plasmin activity were investigated. Comparison of pasteurised milk with amicellar casein solution showed that the dissociation of plasmin and caseins on adding EACA and NaCldecreases interference by caseins, but increases inhibition of plasmin with serum-based inhibitorycomponents. The level of detection and repeatability of this method for plasmin activity analysis wereimproved compared with previous spectrophotometric assays.
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1. Introduction
The major indigenous proteinase in milk, plasmin (PL; EC3.4.21.7), is the active part of a complex enzyme system. Itszymogen, plasminogen (PLG), is present in raw, bovine milk at a 2e30 times higher concentration than PL (Ismail & Nielsen, 2010), andis activated by two families of plasminogen activators (PA),urokinase-type PA (uPA) and tissue-type PA (tPA). The activity of PLand PA is regulated by plasmin inhibitors (PIs), such as a2-anti-plasmin, and plasminogen activator inhibitors (Grufferty & Fox,1988; Ismail & Nielsen, 2010; Korycka-Dahl, Dumas, Chene, &Martal, 1983; Lu & Nielsen, 1993; Precetti, Oria, & Nielsen, 1997).The level of PL in milk can vary and depends on environmentalfactors, such as stage of lactation and somatic cell count (Larsenet al., 2010; Nicholas, Auldist, Molan, Stelwagen, & Prosser, 2002).
PL readily hydrolyses b- and aS2-casein, and, to a lesser extent, aS1-casein (Grufferty & Fox, 1988). PL activity may be linked to theformation of unclean and bitter off-flavours in milk, and age gela-tion of UHT milk (Harwalkar, Cholette, McKellar, & Emmons, 1993;Kelly & Foley, 1997). On the other hand, hydrolysis of caseins by PLplays an important role in the initial ripening of Swiss cheese types(Bastian & Brown, 1996).
Various methods to measure PL and PLG-derived activities havebeen described (Politis, Zavision, Barbano, & Gorewit, 1993;Richardson & Pearce, 1981; Rollema, Visser, & Poll, 1983; Saint-Denis, Humbert, & Gaillard, 2001). The determination of PL activ-ity in these assays is based on the hydrolysis of a specific substrate,which upon cleavage releases a chromogenic (Rollema et al., 1983)or fluorogenic product (Richardson & Pearce, 1981; Saint-Deniset al., 2001). The different sample preparations used in these as-says, however, cause varying results and different interfering ef-fects have been reported. Two substrates (S-2251 and SpectrozymePL) have been reported to enhance PLG activation (Kolev, Owen, &Machovich, 1995), while ε-aminocaproic acid (EACA), a lysine
* Corresponding author. Tel.: þ45 87332783.E-mail address: [email protected] (V.M. Rauh).
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International Dairy Journal 38 (2014) 74e80
derivative often used to enhance PL activity, inhibits activation ofhuman PLG (Alkjaersig, Fletcher, & Sherry, 1959; Cesarman-Maus &Hajjar, 2005). EACA dissociates PL and PLG from caseins by bindingto the lysine-binding sites on PL and PLG (Korycka-Dahl et al., 1983).
Furthermore, it has been shown that the dissociation of humanPLG by EACA also inhibits activation of PLG by tPA and uPA(Cesarman-Maus & Hajjar, 2005; Collen, 1987; Sun et al., 2002). Onthe other hand, the natural substrate of PL in milk, the caseins, canact as competitive inhibitors towards a chromogenic substrate usedin the assays (Bastian, Brown, & Ernstrom, 1991a) and affect theactivation of PLG (Heegaard, Andreasen, Petersen, & Rasmussen,1997; Markus, Hitt, Harvey, & Tritsch, 1993). Whey proteins arealso able to inhibit PL activity (Hayes, McSweeney, & Kelly, 2002;Politis et al., 1993), but the exact mechanism has not beendescribed so far; b-lactoglobulin and bovine serum albumin haveshown a higher inhibitory effect than a-lactalbumin (Politis et al.,1993).
Another consideration in terms of assay suitability and perfor-mance is that, especially for fluorescence assays, the turbidity ofmilk can severely interfere with the measurement. In most assaysdescribed, an extensive sample preparation protocol is needed toavoid these interferences (Politis et al., 1993; Rollema et al., 1983).In case of fluorescence methods, a high dilution of the sample(Richardson & Pearce, 1981) or an additional clarification treatment(clarifying reagent�) before measurement (Saint-Denis et al.,2001) is required. These preparations may not only reduce thesensitivity of the assay, but could also further complicate thecomparison of PL activities measured by different sample prepa-rations, substrates and detection methods (Kelly, O’Flaherty, & Fox,2006).
The aim of this study was to combine the spectrophotometricassay for PL and PLG-derived activity described by Rollema et al.(1983) with the sample preparation proposed by Saint-Deniset al. (2001) and expand it to turbid, fat-containing milk typesand different sample matrices including cheeses. A further aimwasto study the effect of skimming on PL activity in differentmilk typesand to characterise the inhibitory effects of casein and serum-based components on the assay, especially the effect of dissocia-tion of PL and caseins by EACA and NaCl on PL and PLG-derivedactivity in different sample types.
2. Materials and methods
2.1. Materials
PL from bovine serum was obtained from Roche Diagnostics(Hvidovre, Denmark) and urokinase (Thrombolysin) was fromImmuno Danmark A/S (Copenhagen, Denmark). The ε-amino-caproic acid (EACA) was obtained from SigmaeAldrich DenmarkApS (Brøndby, Denmark) and H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251) from Haemochrom Diagnostica GmbH(Essen, Germany).
2.2. Origin of samples
The pasteurised (minimum of 72 �C for 15 s) skim milk (SM),homogenised and pasteurised whole milk with 3.5% fat (WM), andUHT milk (0.5% fat) used in this study were commercially availablemilk products obtained from a local supermarket, stored at 5 �C andanalysed before expiry date. Raw bulk milk (RM) was obtained fromArla Foods (Brabrand Dairy Plant, Brabrand, Denmark). A whey-protein-free milk serum (UF-permeate) was prepared by ultrafiltra-tion of SM using 5-kDa spiral wound membranes (Koch MembraneSystems HFK-328). A micellar casein solution (MCS) was prepared byultracentrifugation of SM at 100,000 � g for 60 min at 25 �C. The
casein pellet was reconstituted with UF permeate to original volumeand gently stirred at 4 �C overnight. Samples of Swedish Herrgårdcheese (>28% fat,>6months)andparmigianoReggiano (>32% fat,>6months) were obtained from a local supermarket. Danish Havarticheese (>60% fat, 1 week) was produced in the pilot plant at ArlaFoods Strategic Innovation Center (Brabrand, Denmark).
2.3. Basic sample preparation and measurement of PL and PLG-derived activity
Milk samples (1 mL) were mixed with 250 mL 0.4 M tri-sodiumcitrate buffer, pH 8.9, and shaken for 15 min to dissociate thecasein micelles. Sample preparation for cheese was adapted fromthat of Richardson and Pearce (1981) with modifications as follows.A mass of 5 g of grated cheese was dissolved in 45 mL 0.4 M tri-sodium citrate buffer, pH 8.9, and stirred for 30 min at room tem-perature, followed by holding for 30 min at 45 �C, and finally,15 min at room temperature to separate the fat phase. The solutionwas then centrifuged at 7500 � g for 15 min, and the supernatantbelow the cream phase was removed and centrifuged again underthe same conditions.
The citrate-treated milk or cheese samples were diluted 1:1 (v/v) with an assay buffer as described by Saint-Denis et al. (2001)containing 0.1 M TriseHCl, 8 mM EACA, 0.4 M NaCl, pH 8, andmixed for 15 min to dissociate PL and PLG from the caseins.
For determination of PLG-derived activity, PLG in the citrate-treated samples was activated by a 1:1 (v/v) dilution with anassay buffer containing 200 Plough units mL�1 urokinase andincubated for 60 min at 37 �C (Korycka-Dahl et al., 1983; Rollemaet al., 1983; Saint-Denis et al., 2001).
PL and PLG-derived activity were determined by measuring therate of hydrolysis of the chromogenic substrate S-2251 (Rollemaet al., 1983) and release of p-nitroaniline. To compensate forlower dilution of 1:5 v/v the sample in the final mixture comparedwith 1:10 (v/v) in the assay described by Rollema et al. (1983) and,to increase the sensitivity of the assay, the substrate concentrationwas increased from 0.6 mM S-2251 (Rollema et al., 1983) to 2 mM inthe final mixture. The substrate, S-2251, was dissolved in 0.1 M TriseHCl buffer, pH 8 to a concentration of 4 mM.
The sample solution (50 mL) was transferred to a microtitre plateand the reaction was initiated by addition of 50 mL of the substratesolution. Absorbancewas read at 405 nm and 490 nm at 37 �C usingan ELISA plate reader (Bio-Tek EL 808 and Bio-Tek Synergy Mx,BioTek Instruments Inc., Wisconsin, IL, USA) at intervals of 2e10 min for 60e180 min depending on the level of PL activity in thesample. Substrate volumes per sample were minimised throughuse of Costar 3695 clear half-area polystyrene 96-well plates(Corning Inc., New York, NY, USA). To correct for turbidity, thebackground absorbance values (490 nm) were subtracted from theabsorbance values at 405 nm, corresponding to colour develop-ment due to release of p-nitroaniline. Correction for turbidity usingthe absorbances at 540 and 630 nmwas also tested. The increase inabsorbance, indicated as DA405 nme490 nm as a function of time, dA/dt, was converted into PL units using a standard curve preparedwith commercial PL solution from a PL stock solution (0.5 U in Trisbuffer, pH 8) with assay buffer additionally containing 4 mg mL�1
gelatine to stabilise PL. The correlation between PL activity andabsorbance was linear in the range 15 mU mL�1 to 1 mU mL�1
(R2¼ 0.999). No increase over time in absorbancewas observed in ablank sample containing assay buffer with 4mgmL�1 gelatine only.
2.4. Method validation
The linearity of the assaywas evaluated bymeasuring PL activityin undiluted SM as well as 1:1 and 1:10 (v/v) dilutions of SM and 3
V.M. Rauh et al. / International Dairy Journal 38 (2014) 74e80 75
different wavelength to correct for turbidity (490, 540 and 630 nm).The wavelength (490, 540 or 630 nm) used for turbidity correctionhad no significant effect on the slope, dA/dt, of the curves, forreference, 490 nm was thus selected. Linearity and turbidity of theassay for fat containing milks was assessed withWM and RM usingabsorbance at 490 nm to correct for turbidity.
Repeatability of the assay was evaluated by measuring PL andPLG-derived activity of a SM sample for a total of 14 times.Reproducibility was assessed by measuring PL and PLG-derivedactivity in frozen aliquots of SM, RM, Danish Havarti cheese,Swedish Herrgård cheese and Parmigiano Reggiano on 4 occasions(measured on 4 different plates on 4 different days).
Limit of detection was compared with that determined by Saint-Denis et al. (2001) by diluting SMwith 0.1 M TriseHCl, pH 8. The limitof detectionwas the highest dilution of SMwith a significantly higheractivity thanablankconsistingof assaybufferwith4mgmL�1 gelatine.
2.5. Effect of skimming on plasmin activity in milk
The effect of fat removal by centrifugation on the developmentof absorbance in the PL assay was investigated using RM and WMwith SM as control. Prior to skimming, a volume of 2.5 mL 0.4 M tri-sodium citrate buffer, pH 8.9 was added to 10 mL of RM, WM or SMand the solutions were mixed for 15 min. One mL of each solutionwas taken out and stored at 4 �C to ensure the same experimentalconditions, while the remaining volume was further prepared forskimming by centrifugation at 10,000 � g for 10 min at 4 �C. Theupper fat-containing layer was discarded and 1 mL of the clearliquid was used for further analysis. Both the un-skimmed andskimmed samples were further diluted 1:1 (v/v) 0.1 M TriseHCl,8 mM EACA, 0.4 M NaCl, pH 8 and PL activity was measured asdescribed in Section 2.1. Three independent batches of each milk(RM, WM and SM) were used for the experiment and samplepreparation was performed in duplicate.
2.6. Recovery of activity of added PL and inhibitory effects of milkcomponents
Recovered activity of PL was measured by addition of a definedamount of PL with known activity to samples, along with the assaybuffer containing 0.1 M TriseHCl, 8 mM EACA, 0.4 M NaCl, pH 8, toinvestigate possible inhibitory and interfering effects in the assay.Samples tested were RM, SM, UHT milk, MCS as well as Havarti,Herrgård and Parmigiano Reggiano cheese. Furthermore, to providea reference system, which does not contain the PL inhibitors pre-sent in milk, a gelatine solution (4 mg mL�1) containing 0.1 M TriseHCl buffer, pH 8 was prepared. Gelatine has earlier been shown tobe a suitable agent for stabilising PL (Saint-Denis et al., 2001). Themeasured activity of PL in the gelatine system was set as 100%recovered activity.
The effect of sample concentration on recovered activity andinhibitory effects was evaluated by diluting a UHTmilk samplewith0.1 M TriseHCl buffer, pH 8, to a final concentration of UHT milk of100, 75, 50 and 25% (v/v) and a constant amount of added PL. PLactivity in the UHT milk dilution was compared with PL activity indilutions of a SM sample to which UHT milk had been added to afinal proportion of SM in the mixture of 100, 75, 50 and 25% (v/v).PL activity was measured as described in Section 2.3.
2.7. Effect of dissociation of plasmin by EACA and NaCl in presenceand absence of whey proteins on PL and PLG-derived activity
To further investigate the effect of milk components, PL wasdissociated from casein by the use of EACA and NaCl (EACAþ NaCl).EACA þ NaCl at concentrations of 4 mM and 2 M, respectively, have
been shown previously to dissociate PL from casein micelles (Saint-Denis et al., 2001).
The effect of EACAþNaCl on PL and PLG-derived activities in RMand SM was evaluated by diluting the sodium-citrate-treatedsamples (1:1 v/v) with either 0.1 M TriseHCl, pH 8, or 0.1 M TriseHCl, pH 8, containing 8 mM EACA and 0.4 M NaCl.
The effect of pre-incubationwith EACAþNaCl on PL activity wascompared for a SM and a MCS sample prepared from the same SM.A dilution series of SM and MCS was prepared by addition of 0.1 M
TriseHCl buffer, pH 8, to a final concentration of SM or MCS of 100,80, 60, 40 and 20% (v/v). From each of these dilutions, 1 mL wastaken and a volume of 250 mL 0.4 M tri-sodium citrate buffer, pH 8.9was added. Each sample was shaken for 15 min before furtherdilution (1:1, v/v) with either 0.1 M TriseHCl, pH 8, or 0.1 M TriseHCl, pH 8, containing 8 mM EACA and 0.4 M NaCl. PL activity wasmeasured as described in Section 2.3.
2.8. Statistical analysis
Analysis of variance (ANOVA) and separation of means wascarried out using MATLAB (Mathworks, Natick, MA, USA). One-way-ANOVA was used to determine significant differences(P� 0.05) among groups. Differences between themeans andmeanseparation were determined using TukeyeKramer multiple meanscomparison tests (P � 0.05 and 0.01).
3. Results and discussion
3.1. Method validation
The linearity of the assaywas evaluated bymeasuring PL activityin undiluted SM as well as 1:1 and 1:10 (v/v) dilutions of SM with0.1 M TriseHCl buffer, pH 8. The reference wavelength measure-ments showed that the turbidity in undiluted and diluted SMsamples was not constant throughout the assay. Within the first20 min, the turbidity decreased rapidly in an exponential manner,which led to a non-linear increase in the DA405 nme490 nm in thattime period (Fig. 1), and this increase appeared to be independentof sample concentration. The first 20 min of the measurementswere therefore excluded from the calculations of the slope.Following this, the absorbance, DA405 nme490 nm increased linearly(20e120 min) in both the diluted and undiluted SM samples(R2 > 0.99).
Fig. 1. Plasmin activity measured as development in absorbance DA405 nme490 nm dueto substrate hydrolysis and release of 4-nitroaniline in undiluted (B), 1:1 (,) and 1:10(7) dilution of pasteurised skim milk with 0.1 M TriseHCl buffer, pH 8 (average of 3determinations � standard deviation). Lines are linear fits for the time period 20e120 min to guide the eye.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 74e8076
The development of absorbance at 405 nm and at 490 nm in RM,WM and SM are shown in Fig. 2A. The assay with RM was onlystable up to 2 h, after which time creaming occurred, and no furthermeasurement was possible. Due to the presence of fat, the initialabsorbance at 405 and 490 nm of RM and WM was 4e5 timeshigher compared with SM. The absorbance at 405 nm in SM andWM increased slightly during the incubation time, but decreased inRM, which may be due to the onset of creaming. At the same time,the absorbance at 490 nm decreased for both RM and SM, while itremained almost constant for SM. Fig. 2B shows the developmentin absorbance DA405 nme490 nm after correction for turbidity. As canbe seen, the absorbance increased linearly in all milk samples, andthere was only a minor difference in the initial absorbance. Thecorrection for turbidity using a reference wavelength at 490 nmthus enables measurements in turbid samples.
Reproducibility of the assay and performance in different samplematrices was assessed by measurement of PL and PLG-derived ac-tivity in frozen aliquots of RM, SM,1weekoldDanishHavarti cheese,Herrgård cheese and Parmigiano Reggiano on 4 occasions (Table 1).SM showed a higher PL activity than RM, while the PLG-derivedactivity was similar in SM and RM. The PLG/PL ratio was 8.2 for RMand 7.1 for SM, which is in the reported range for milk (Ismail &Nielsen, 2010). Due to the fat content, the reproducibility in RMwas poorer comparedwith SM,with a relative standard deviation of12.1% for PL activity and 4.1% for PLG-derived activity, comparedwith 2.8% and 1.1% for SM. PLG-derived activity in theHavarti cheesewas approximately 11.5 times higher than the PL activity and wasapproximately 2 times higher than the PL activity in the Herrgårdcheese (Table 1). The PLG to PL ratio of the Havarti cheese is withinthe range of previously reported values of approximately 3(Benfeldt, 2006) to 21 (Bastian, Hansen, & Brown, 1991b).
To our knowledge, PL and PLG-derived activity in Herrgårdcheese has not been described previously. PLG-derived activity inParmigiano Reggiano was similar to that in the Herrgård cheese,while PL activity was lower. The relative standard deviations of thePL and PLG-derived activity in the cheeses (Table 1) were from 3.0to 4.1% for PL activity and from 1.3 to 3.7% for PLG-derived activity.In comparison, Richardson and Pearce (1981) reported a repro-ducibility of 9% for a Swiss-type cheese (n ¼ 9). Repeatability of theassay was assessed by measuring a SM sample 14 times. Therepeatability, measured as % relative standard deviation, for PL andPLG-derived activity was 2% and 1%, respectively, compared with2% reported for PL activity by Saint-Denis et al. (2001) and 10%reported for PL activity by Rollema et al. (1983).
The lowest PL activity significantly higher than a blank sample(P< 0.05)was detected in a 100-fold diluted sample of SM, indicatingan overall limit of detection of 1%. This is an improvement compared
with previous chromogenic methods, where the level of detectionwas reported by Rollema et al. (1983) to be between 6 and 10% of PLactivities in bulk milk, but does not reach the level of detection offluorogenic methods (0.1% according to Saint-Denis et al., 2001).
3.2. Effect of skimming by centrifugation on plasmin activity in milk
Previous methods for measurement of PL activity have beenbased on measurements in skimmed milk (Rollema et al., 1983),and have either included a centrifugation step to remove the creamphase (Politis et al., 1993; Richardson & Pearce, 1981) or do notspecify the fat content of the milk tested (Saint-Denis et al., 2001).To investigate the effect of skimming on the development ofabsorbance in the assay, samples of WM and RM were analysedbefore and after skimming, with SM as a control.
Table 2 shows the measured PL activities before and after skim-ming and the percentage difference in PL activity due to skimming.The PL activity in un-skimmedWMwasw25%higher comparedwiththe SM, but decreased significantly by almost 30% after skimming(P � 0.01), to a similar level as in the SM, while skimming had nosignificant effect on the PL activity in RM and SM (P � 0.05). Themeasurements in RM provide further proof that the describedmethod is suitable formeasurement in turbid fat-containing samples.
The decrease in PL activity in WM after skimming could beattributed to the removal of PL associated with caseins on the milkfat globules. The native milk fat globule membrane fraction of rawmilk contains a low level of PL activity, probably due to caseincontamination (Benfeldt, Larsen, Rasmussen, Andreasen, &Petersen, 1995; Politis, Barbano, & Gorewit, 1992). Upon homoge-nisation, the composition of the milk fat globule membrane(MFGM) changes and up to 70% of the reformedmembrane consistsof caseins and casein micellar fragments (Cano-Ruiz & Richter,1997). The high initial PL activity and the decrease in PL activityin theWMupon skimming indicates that a large portion of PL couldbe associated with the caseins in the MFGM after homogenisation
Fig. 2. Development of (A) absorbance at 405 (open symbols) and 490 nm (filled symbols) in raw milk (B,C), whole pasteurised milk (,,-) and pasteurised skim milk (>,A) andof (B) DA405 nme490 nm.
Table 1Plasmin and plasminogen-derived activity (mU mL�1) in milk and cheese samples.a
Sample Plasmin activity Plasminogen-derivedactivity
Raw milk 390 � 47 3203 � 130Pasteurised skim milk 462 � 13 3259 � 37Havarti (1 week old) 4095 � 167 46,940 � 600Herrgård (>6 months) 10,225 � 309 25,841 � 958Parmigiano Reggiano 8579 � 228 25,728 � 255
a Results are shown as means � standard deviation of 4 determinations on 4occasions. PL and PLG derived activity are shown in mU mL�1 for milk and in mU g�1
for cheeses.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 74e80 77
and explain the similar PL activities of the skimmed WM and SM.The higher PL activity in WM compared with SM is in contrast toearlier findings, which reported that 40% of PL is inactivated duringa standard single-stage homogenisation of milk (Hayes & Kelly,2003). Initial experiments to confirm PL activity in the reformedMFGM of homogenised creamwere not possible due to difficulty ofdispersing of the washed cream in the presently used buffer sys-tems. In conclusion, skimming and centrifugation of the sampleprior to analysis facilitates the measurement of PL in RM samples,but leads to an underestimation of the PL activity in homogenisedmilk.
3.3. Recovery of activity of added PL and inhibitory effects of milkcomponents
Fig. 3A shows the PL activity in a dilution of SM with UHT milkand the activity of a fixed amount of PL added to a dilution of UHTwith buffer. PL activity in the dilution of SM with UHT milkdecreased linearly with increasing proportion of UHT milk addedwith the same casein and whey protein concentration. Loweringthe whey protein and casein concentration increased measured PLactivity, as can be seen from the increase in activity of the added PLin UHT milk with decreasing proportion of added UHT milk. Therecovered activity of the added PL in UHT milk increased from48.1�1.3% in the sample containing UHTmilk only to 71.2� 0.7% inthe dilution containing 25% UHT milk (Fig. 3B), which shows theinhibitory and interfering effects of caseins and denatured wheyproteins (Bastian, Hansen, & Brown,1993; Hayes et al., 2002) on thePL activity.
The recovered activity of added PL in SM, RM, MCS and UHTmilk, and Danish Havarti, Swedish Herrgård and ParmigianoReggiano cheese, are shown in Table 3. The recovered activity in themilk samples was correlated with the heat treatment of the sam-ples with RM showing the lowest recovered activity and UHT milkthe highest. The increase in recovered activity with increasing heattreatment is most likely due to inactivation of PI, which has beenshown to be heat-labile (Prado, Sombers, Ismail, & Hayes, 2006).The recovery rate in MCS was only approximately 67%, due tocompetitive inhibition of the synthetic substrate by caseins (Bastianet al., 1991a). Compared with the milk samples, MCS showed a 16e29% higher recovered activity, reflecting the inhibitory effect ofserum-based PI and whey proteins on PL activity.
Bastian et al. (1991a) showed that activity of PL towards S-2251is competitively inhibited by caseins and, by increasing the sub-strate concentration to 0.4 mM, eliminated the inhibitory effect of5 mg mL�1 casein. In this assay, a casein concentration of approx-imately 2.6 mg mL�1 in the MCS and >4 times higher substrateconcentration compared with those used by Bastian et al. (1991a)had an inhibitory effect on the PL activity. The differences inthese results may arise from the source of PL and higher PL con-centrations used for the experiment. In this study, the activity of theindigenous PL associated with the casein fraction was measured,whereas Bastian et al. (1991a) used commercial PL from bovineplasma (final concentration 26.8 nM) at a concentration 3e17 timeshigher than that present in milk (1.6e8.2 nM).
Recovered activity in cheeses was similar to that for MCS,although the protein concentration in the final mixture wasapproximately double that of the milk samples. Protein content inthe cheeses varied due to the removal of fat and the recoveredactivity in the cheese samples increased with increasing fat con-tent. Although the fat content in the Danish Havarti cheese wasapproximately twice as high as for the other cheese types, therecovered activity was only slightly higher. The Havarti cheese wasonly oneweek old and the hydrolysis of caseins during the ripening
Fig. 3. PL activity (A) in pasteurised skim milk diluted with UHT milk without detectable PL activity (B) and activity of added PL in UHT milk (0.5% fat; >) diluted with 0.1 M TriseHCl buffer, pH 8; percentage (B) of recovered activity of added PL to a dilution series from 0 to 100% of commercial UHT milk with 0.1 M TriseHCl buffer, pH 8. Results are shown asaverage of 3 determinations � standard deviation.
Table 3Recovered activity of added PL in different milk and cheese types.a
Sample Recovered activity (%)
Raw milk 38.8 � 2.9a
Pasteurised skim milk 43.8 � 5.3b
UHT milk 50.3 � 2.7c
Micellar casein 66.9 � 1.5d
Havarti 70.4 � 1.8a
Herrgård 64.8 � 0.8b
Parmigiano Reggiano 65.5 � 3.3b
a Results are shown as means of 3 determinations � standard deviation;values without common superscripts are significantly different (P � 0.05).
Table 2PL activity in three independent batches (A-C) of pasteurised, homogenised wholemilk with 3.5% fat (WM), raw bulk milk (RM) and pasteurised skimmilk (SM) beforeand after skimming by centrifugation.a
Milk sample Before skimming(mU mL�1)
After skimming(mU mL�1)
Percentage activityafter skimming
WMA 703 � 33 509 � 10 72.4 � 5.1B 683 � 35 491 � 5 71.9 � 5.2C 679 � 24 482 � 2 70.9 � 3.6
RMA 558 � 6 561 � 3 100.4 � 1.1B 636 � 9 694 � 3 109.2 � 1.9C 712 � 18 646 � 10 90.8 � 2.9
SMA 549 � 15 523 � 8 95.1 � 3.2B 534 � 8 552 � 9 103.3 � 2.1C 558 � 15 563 � 1 101.0 � 2.7
a Results are means of 2 determinations � standard deviation.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 74e8078
of the cheeses could cause a reduced inhibitory effect on the syn-thetic substrate as they could be too small to block the active centreor not have a cleavage site for PL and thus not bind at all. Hydro-lysed caseins and the resulting peptides may have a reducedinhibitory effect on the synthetic substrate.
The recovered activities for milk and micellar casein in Table 3are in the same range as previously reported results. Saint-Deniset al. (2001) reported a recovery of 30e40% in diluted (1:4, v/v)pasteurised milk, while Richardson and Pearce (1981) reported arecovery of 40% in a 1:16 (v/v) diluted 3% solution of sodiumcaseinate. This shows that the described method has a similarsensitivity and sufficient substrate concentration to compensatefor inhibitory effects compared with previously describedmethods, although a direct comparison is difficult due to thedifferent buffers, dilutions and samples used for thedetermination.
3.4. Effect of dissociation of plasmin by EACA and NaCl in thepresence and absence of whey proteins on PL and PLG-derivedactivity
PL in milk is known to be associated with caseins via lysine-binding sites that are not affected by addition of trisodium citrate(Politis et al., 1993; Rollema et al., 1983). EACA has previously beenshown to dissociate PL and PLG from caseins by binding to thelysine-binding sites of PL and PLG, and enhance measured PL ac-tivity (Korycka-Dahl et al., 1983). The lysine-binding sites of PL arealso of great importance for the physiological function of human PLin blood and the fibrinolytic system. Binding of PL to fibrin ensuresa close proximity of the enzyme to its substrate and also protects PLfrom irreversible inhibition by a2-antiplasmin, a major indigenousPL inhibitor, which requires a free lysine-binding site and a freecatalytic centre of PL to interact and inhibit PL (Cesarman-Maus &Hajjar, 2005; Collen, 1987). The dissociation of PL and caseinscould therefore reduce the competitive inhibition of the syntheticsubstrate by caseins and affect the interaction of PL with PI andwhey proteins.
The dissociation of PL and caseins by EACA þ NaCl increased PLactivity in SM significantly (P > 0.05) by approximately 9% and 11%for PL and PLG-derived activity, respectively (Table 4). The PLG/PLratio of the free and bound SM was not significantly different,indicating that EACA þ NaCl did not affect the activation of PLG byuPA under the chosen assay conditions. The dissociation of PL didnot lead to a significant increase in PL activity in RM, suggestingthat the dissociation increased inhibition of PL by serum-basedinhibitors.
To further investigate the inhibitory effect of serum-basedinhibitory components on free and bound PL, EACA and NaClwere added together to a dilution series of SM and MCS. Fig. 4shows measured PL activity as a function of the percentage of SMor MCS in the dilutions in the presence or absence of EACA þ NaCl.
The PL activity in the MCS did not decrease proportionally withthe casein content. This suggests that the inhibitory effect of caseinincreases with concentration, as previously reported by Bastianet al. (1991a). Dissociation of PL and caseins in the undiluted MCSincreased PL activity by 44%. This shows that the dissociation of PLand caseins greatly reduces the competitive inhibition of the syn-thetic substrate by caseins in the assay. The difference in PL activitybetween free and bound PL in MCS was constant in all dilutions,indicating that the same competitive inhibition applies at differentcasein concentrations.
Compared with MCS, the increase in PL activity upon dissoci-ation of PL and caseins in SM was less pronounced. In contrast tothe MCS, the difference in PL activity increased with increasingdilution from 9% in the undiluted SM to 24% in the sample
containing 20% SM, showing that dilution of serum-based in-hibitors increases PL activity. For the SM samples, on the otherhand, the relatively small difference in PL activity compared withthe MCS samples indicates that the interfering effect of the caseinsis overshadowed by a change in the interaction of PL with serum-based inhibitory components (e.g., whey proteins). Withincreasing sample dilution, the percentage difference betweensamples with free and bound PL increased from an initial of 8% inthe 100% SM to 19% in the 20% SM. This shows that the inhibitoryeffect of serum-based components decreases with increasingdilution of SM.
Since the interaction of PL and a2-antiplasmin is reduced inpresence of EACA (Christensen, Sottrup-Jensen, & Christensen,1995) and pasteurisation conditions largely inactivate PIs (Pradoet al., 2006), the observed inhibition is most likely due to anenhanced inhibition of PL bywhey proteins. This protective effect ofcaseins towards inhibition of PL by whey proteins in milk has not toour knowledge been documented previously, although this wassuggested by Politis et al. (1993). Whether the dissociation of PLand caseins also enhances the inhibition of PL in a native milkenvironment and reduces proteolysis of casein was not the focus ofthis study, but should be further investigated to allow a correlationof proteolysis and PL activity.
Table 4Effect of dissociation of PL and caseins by EACA plus NaCl on PL and PLG derivedactivity in skimmed raw milk and pasteurised skim milk.a
Sample Plasminactivity(mU mL�1)
Plasminogen-derivedactivity (mU mL�1)
Plasminogen/plasminratio
Raw milk þ EACAand NaCl
438 � 4a n.d.
Raw milk � EACAand NaCl
443 � 6a n.d.
Pasteurised skimmilk þ EACAand NaCl
459 � 6b 3367 � 87a 7.3 � 0.2a
Pasteurised skimmilk � EACAand NaCl
422 � 6c 3036 � 47b 7.2 � 0.2a
a Results are shown as mean 3 determinations � standard deviation (n.d., notdetermined); values without common superscripts for each row are significantlydifferent (P < 0.05).
Fig. 4. Effect of adding EACA þ NaCl on the plasmin activity in SM (,,-) and amicellar casein solution (B,C). Milk and the micellar casein solution were dilutedwith TriseHCl buffer, pH 8. The samples were mixed 1:1 with TriseHCl buffer without(dashed line, filled symbols) or with 6 mM EACA and 0.4 M NaCl (solid line, opensymbols) and assayed for plasmin activity (average of 3 determinations � standarddeviation).
V.M. Rauh et al. / International Dairy Journal 38 (2014) 74e80 79
4. Conclusion
The method presented enables the measurement of PL and PLG-derived activity in turbid samples, such as homogenised wholemilk, micellar casein solutions and cheese extracts, using a micro-titre plate real-time kinetic method with high substrate. The per-formance of the assay was determined in relation to limit ofdetection, repeatability, reproducibility, recovery of added PL andthe effect of milk concentration on the linearity of the assay. Whenpasteurised, homogenised milk was centrifuged, approximately30% of the PL activity was removed, in contrast to raw, whole milk,where the level did not decrease after skimming. The dissociationof PL from the caseins by simultaneous addition of EACA and NaClmainly affected interactions of PL with inhibitory componentspresent in whey, e.g., PIs and whey proteins, which reduced PLactivity by approximately 30e40%. Further studies will be requiredto clarify the effect of homogenisation on the PL system and PLactivity and the mechanism of inhibition of PL by whey proteins.
Acknowledgements
The Danish Agency for Science, Technology and Innovation(DASTI) is thanked for financial support of the study. We thankVibeke Mortensen from Arla Foods Strategic Innovation Centre forexcellent technical assistance and Prof. Jacob Holm Nielsen (Uni-versity of Copenhagen) for helpful discussions.
References
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Hayes, M. G., & Kelly, A. L. (2003). High pressure homogenisation of milk (b) effectson indigenous enzymatic activity. Journal of Dairy Research, 70, 307e313.
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Heegaard, C. W., Andreasen, P. A., Petersen, T. E., & Rasmussen, L. K. (1997). Bindingof plasminogen and tissue-type plasminogen activator to dimeric as2-caseinaccelerates plasmin generation. Fibrinolysis and Proteolysis, 11, 29e36.
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milk. Journal of Dairy Science, 66, 704e711.Larsen, L. B., Hinz, K., Jørgensen, A. L. W., Møller, H. S., Wellnitz, O.,
Bruckmaier, R. M., et al. (2010). Proteomic and peptidomic study of proteolysisin quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus.Journal of Dairy Science, 93, 5613e5626.
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Politis, I., Zavizion, B., Barbano, D. M., & Gorewit, R. C. (1993). Enzymatic assay forthe combined determination of plasmin plus plasminogen in milk: Revisited.Journal of Dairy Science, 76, 1260e1267.
Prado, B. M., Sombers, S. E., Ismail, B., & Hayes, K. D. (2006). Effect of heat treatmenton the activity of inhibitors of plasmin and plasminogen activators in milk.International Dairy Journal, 16, 593e599.
Precetti, A. S., Oria, M. P., & Nielsen, S. S. (1997). Presence in bovine milk of twoprotease inhibitors of the plasmin system. Journal of Dairy Science, 80, 1490e1496.
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V.M. Rauh et al. / International Dairy Journal 38 (2014) 74e8080
Paper II
Rauh, V. M., Sundgren, A., Bakman, M., Ipsen, R., Paulsson, M., Larsen, L. B., & Hammershøj,
M. (2014)
Plasmin activity as a possible cause for age gelation in UHT milk produced by direct steam
infusion
International Dairy Journal, 38, 199-207
Plasmin activity as a possible cause for age gelation in UHT milkproduced by direct steam infusion
Valentin M. Rauh a,b,*, Anja Sundgren c, Mette Bakman a, Richard Ipsen d, Marie Paulsson e,Lotte B. Larsen b, Marianne Hammershøj b
aArla Foods Strategic Innovation Centre, Rørdrumvej 2, DK-8220 Brabrand, DenmarkbAarhus University, Dept. of Food Science, Faculty of Science and Technology, Blichers Allé 20, DK-8830 Tjele, DenmarkcArla Foods Strategic Innovation Centre, Lindhagensgatan 126, SE-10546 Stockholm, SwedendUniversity of Copenhagen, Department of Food Science, Rolighedsvej 30, DK-1958 Frederiksberg C, DenmarkeDept of Food Technology, Engineering and Nutrition, Lund University, SE-22100 Lund, Sweden
a r t i c l e i n f o
Article history:Received 20 September 2013Received in revised form18 December 2013Accepted 18 December 2013Available online 8 January 2014
a b s t r a c t
The effect of enzymatic activity in direct steam infusion heat treated milk with ultra-short holding times(>150 �C for <0.2 s) on age gelation during storage was investigated. Preheating at either 72 or 95 �C for180 s was performed. Milk pre-heated at 72 �C showed extensive proteolysis and exhibited bitter offflavour and contained <40% intact aS- and b-caseins after 6 weeks storage at 20 �C. No proteolysis of k-casein was detected. Plasmin was identified as active protease and activation of plasminogen wasobserved as an increase in the rate of casein hydrolysis. Proteolysis in the stored samples correlated witha decrease in pH and with changes in colour. Gelation occurred after 10 weeks along with an increase inviscosity and extensive proteolysis of aS- and b-caseins. In conclusion, plasmin activity was involved inage gelation and bitterness caused by proteolysis was the shelf-life limiting factor.
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1. Introduction
Ultra-high temperature (UHT) treatment of milk is a heatingprocess at very high temperatures for short holding times, whichrenders the milk commercially sterile and gives a product with along shelf life at ambient temperatures. UHT treatments are usuallycarried out at temperatures of 140 �C for 4e6 s. Both indirect anddirect heating systems (steam injection or infusion) have beenapplied to increase the shelf-life of milk. More recently, UHTtreatments with temperatures >150 �C and ultra-short holdingtimes (<0.2 s) have been shown to result in a sufficient reduction ofmicro-organisms, while doing minimal heat damage to milk con-stituents, such as vitamins and whey proteins (De Jong, 1996).
A major limiting factor for the shelf life of UHT milk is proteol-ysis of caseins, which can be caused by residual heat-stable bac-terial proteases, indigenous milk proteases or combinations ofthese. Such proteolysis can cause formation of bitter off-flavours,sedimentation and age gelation of UHT milk (Datta & Deeth,2003; Kelly & Foley, 1997). These problems more often occur indirectly heated UHT milk, since the heat load is reduced, when
compared with indirect methods, due to the rapid heating andcooling of the milk (Newstead, Paterson, Anema, Coker, & Wewala,2006). This may be solved by inclusion of a preheat treatment step(Newstead et al., 2006; Van Asselt, Sweere, Rollema, & de Jong,2008) or by increasing the temperature during UHT treatment(Topçu, Numano�glu, & Saldamlı, 2006).
The cause for age gelation in UHT milk is not known, but it hasbeen suggested that release of the b-lactoglobulin-k-casein com-plex from the casein micelle is involved (McMahon, 1996). Thiscomplex is formed during heat treatment by the denaturation of b-lactoglobulin (b-lg) and subsequent aggregation with k-casein viadisulphide bonds. The released complex aggregates and forms a gel.Proteolysis accelerates or can cause the release of the b-lactoglob-ulin-k-casein complex by hydrolysis of the caseins that anchor theb-lactoglobulin-k-casein in the caseinmicelle (Datta & Deeth, 2001;McMahon, 1996). While a certain level of proteolysis is required toinduce age gelation, the relation between gelation time and extentof proteolysis is controversial (Manji & Kakuda, 1988; Newsteadet al., 2006).
Plasmin (PL; EC 3.4.21.7), the major indigenous protease in milk,has been closely linked to the age gelation of UHT milk (Kelly &Foley, 1997; Kohlmann, Nielsen, & Ladisch, 1991; Manji & Kakuda,1988; Newstead et al., 2006). PL readily hydrolyses b- and aS2-casein, and to a lesser extent, aS1-casein (Grufferty & Fox,1988). The
* Corresponding author. Tel.: þ45 87332783.E-mail address: [email protected] (V.M. Rauh).
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International Dairy Journal 38 (2014) 199e207
level and activity of PL in milk can vary and depends on biologicalfactors, such as stage of lactation and somatic cell count (Larsenet al., 2010; Nicholas, Auldist, Molan, Stelwagen, & Prosser, 2002).
PL is the active part of a complex enzyme system. Its zymogen,plasminogen (PLG), is present in raw, bovine milk at a 2e30 timeshigher concentration than PL (Ismail & Nielsen, 2010; Korycka-Dahl, Dumas, Chene, & Martal, 1983; Rauh et al., submitted forpublication), and is activated by two families of plasminogen acti-vators (PA), urokinase-type PA (uPA) and tissue-type PA (tPA). Theactivity of PL and PA is regulated by plasmin inhibitors (PIs), such asa2-antiplasmin, and plasminogen activator inhibitors (PAIs)(Grufferty & Fox, 1988; Ismail & Nielsen, 2010; Lu & Nielsen, 1993;Precetti, Oria, & Nielsen, 1997).
The components of the PL system show very different thermalstabilities. While PL, PLG and PA are heat-stable enzymes, the in-hibitors of the PL system are generally considered as heat labile(Prado, Ismail, Ramos, & Hayes, 2007; Prado, Sombers, Ismail, &Hayes, 2006; Richardson, 1983). Due to this complexity it is diffi-cult to predict the actual PL activity and the consequence of pro-teolysis for heat treatment of milk under novel and untested time-temperature conditions.
The aim of the present study was to investigate the effect ofplasmin activity on proteolysis and physical-chemical changes inmilk treated with direct steam infusion heating and ultra-shortholding times during subsequent storage.
2. Materials and methods
2.1. Materials
Onedayold lowpasteurisedmilk (72 �C for 15 s)with 1.5% fatwasobtained fromArla Foods StockholmDairy (Stockholm, Sweden) andprocessed in the UHT pilot plant (SPX, Silkeborg, Denmark) at ArlaStrategic Innovation Centre (Stockholm, Sweden). The milk (200 L)was pre-heated at 72 �C (Milk72) or 95 �C (Milk95) for 180 s in atubular heat exchanger before being subjected to the direct steaminfusion heat treatment (>150 �C for <0.2 s). Pre-heat treatmentconditions were chosen to obtain a milk with (Milk72) and without(Milk95) residual PL activity. After flash cooling, the milk washomogenised using a two-stage homogeniser (160/40 bar), furthercooled down to 20 �C by a tubular heat exchanger and packedaseptically in 300mL glass bottles. The trialswere performed duringthree consecutiveweeks. Themilkwas stored in thedarkat 20 �C in aclimate chamber for 14e16 weeks. Samples, i.e., one 300 mL bottleper treatment, were collected once every week for further analysis.
2.2. Analytical methods
Each batch of processedmilk was tested for sterility according toISO 4833 (ISO, 2003). The milk was visually inspected for sedi-mentation, gelation and colour. Milk72, Milk95 and fresh low pas-teurisedmilk were tasted for perceived bitterness by five panellists.ThepHof themilkwasmeasuredat roomtemperaturebyapHmeter(Knick ElektronischeMessgeräte GmbH& Co. KG., Berlin, Germany).Measurements were performed in duplicates. Colour developmentwas measured using Hunters Lab colour space with a colorimeterMinolta CR-A70 (Minolta Camera Co., Ltd, Osaka, Japan). The L-, a-,and b-values reflect lightness (0 ¼ black, 100 ¼ white), redness(�100 ¼ green, 100 ¼ red) and yellowness (�100 ¼ blue,100 ¼ yellow), respectively. The instrument was calibrated with aMinolta standard plate (standard values: Y ¼ 92.4; x ¼ 0.3161;y ¼ 0.3325). Measurements were performed in triplicate. Viscosityof the milk was measured using a double gap concentric cylindergeometry (inside cup diameter 40.0 mm, inside bob diameter40.8 mm, cylinder height 59.5 mm, operating gap 2.0 mm, sample
volume 6.8mL) on a TA Discovery HR-2 rheometer (TA Instruments,NewCastle, DE, USA). Measurementswere performed at 25 �C. Afterthe temperature was reached, a 15 s pre-shearing step was con-ducted before viscosity was recorded for 120 s at a shear rate of100 s�1. Measurements were performed in triplicate.
The volume based particle size distribution of the milk wasobtained with a Master Sizer 3000 (Malvern Instruments Ltd.,Malvern, UK). Distilled, degassed water was used as dispersant. Therefractive index was set to 1.45 for particles and to 1.33 for thedispersant. Milk was added under agitation at 1000 rpm to thedispersion unit until laser obscurationwas in the range of 5.5e8.0%and particle size distributions were recorded. Measurements wereperformed in duplicate.
After 10 weeks storage, visually detectable gel fragments wereplaced on a microscope slide and stained by adding 1 mL of a 0.02%fluorescein-5-isothiocyanate (FITC; Merck, Darmstadt, Germany)solution in acetone for proteins and a 0.02% Nile red (9-diethylamino-5H-benzophenoxazine-5-one; Merck) solution inacetone for fat. After the acetone was fully evaporated, the sampleswere examined using a 40 � and 100 � oil immersion objective ona Leica DMIRE2 inverted confocal laser scanning microscope (LeicaMicrosystems GmbH, Heidelberg, Germany) fitted with an Ar/Krlaser for FITC and a He 543/594 nm laser for Nile red. FITC wasexcited at 485 nm and emission was recorded between 500 and545 nm. Nile red was excited at 543 nm and the emitted signalrecorded from 560 to 700 nm.
PL and PLG derived activity were measured by the rate of hy-drolysis of the chromogenic substrate S-2251 (Rollema, Visser, &Poll, 1983) and release of p-nitroaniline as described previously(Rauh et al., submitted for publication). PLG was activated byaddition of uPA (Immuno Danmark A/S, Copenhagen, Denmark).The rate of increase in absorbance intensity during the assay isproportional to the PL or PLG derived activity.
Analysis of protein composition in the milk was performed asdescribed by Bonfatti, Grigoletto, Cecchinato, Gallo, and Carnier(2008) with the following modifications. A sample of 200 mL ofmilk (w/w) was frozen at �20 �C until further use.
Disulphide bonds were reduced by the addition of 20 mL 1 M
dithiothreitol and 1 mL 100 mM tri-sodium citrate, 6 M urea at 30 �Cfor 60 min. The samples were centrifuged at 9300� g at 5 �C for10 min. From the clear phase, 200 mL were used for the analysis. Avolume of 5 mL was injected into the LCeMS system. The columnwas a BioSuite� C18 PA-B (C18, 3.5 mm; 2.1 mm � 250 mm, Waters,Milford, MA, USA). Buffer A was Milli-Q water with 0.5% (v/v) tri-fluoroacetic acid (TFA) and buffer B acetonitrile with 0.1% (v/v) TFA.Column temperature was 47 �C and a linear gradient from 34.8% to46.5% buffer B from 2 to 16.5 min was applied at a flow rate of0.35 mL min�1 with UV detection at 214 nm. MS detection wasperformed with a quadrupole time-of-flight mass spectrometerwith (Q-TOF-MS; Agilent 6530 Aqurate mass detector, AgilentTechnologies, Santa Clara, CA, USA) and MS scans were continu-ously recorded between a mass to charge ratio (m/z) of 300 and3500. Data analysis was made with Mass Hunter software (AgilentTechnologies). Measurements were performed in duplicate.
For the analysis of pH 4.6 soluble peptides and native wheyproteins, volumes of 20mL of milk were adjusted to pH 4.6 with 1 M
HCl and centrifuged at 11,000� g for 10 min at 4 �C. From the clearsupernatant, 1.5 mL was taken out and frozen at �20 �C untilfurther use. Prior to analysis, 1 mL of the sample was centrifuged at9300� g at 5 �C for 10min and the supernatant used for the analysisby injecting 10e20 mL to the LC system, depending on the peptideconcentration. The column was a Xbridge BEH 300 (C18, 3.5 mm;2.1 mm � 250 mm, Waters) operated at 45 �C. Buffer A was Milli-Qwater with 0.1% (v/v) TFA and buffer B acetonitrile with 0.5% (v/v)TFA. A linear gradient was applied with 100% buffer A from 3 min,
V.M. Rauh et al. / International Dairy Journal 38 (2014) 199e207200
100%e45% buffer A at 45min at a flow rate of 0.3 mLmin�1 with UVdetection at 214 nm. The degree of denaturation of whey proteinswas calculated as the relative difference in the UV peak area of pH4.6 soluble whey proteins before and after heat treatment. Mea-surements were performed in duplicate.
3. Results and discussion
3.1. Influence of heat treatment
The heat treatment rendered the milk sterile as no microbialactivity was detected during storage. The degree of whey proteindenaturation and residual PL and PLG derived activities for Milk72and Milk95 were measured one day after the heat treatment andare shown in Table 1. In comparison with other direct heat treat-ments applying ultra-short holding times reported earlier, Milk72showed a higher degree of denaturation for b-lg. Previously re-ported values for b-lg denaturation were 19e21% at 150e170 �C(Huijs, Van Asselt, Verdurmen, & De Jong, 2004) and 15% and 28% at150 �C (Dickow, 2011) for skim and whole milk, respectively, bothusing a steam injection system. This difference can be explained bythe additional b-lg denaturation occurring during the pre-heattreatment at 72 �C for 180 s amounting to approximately 2e4%(Tolkach & Kulozik, 2007) and additionally approximately 4% dur-ing the homogenisation step following the direct heat treatment(García-Risco, Ramos, & López-Fandiño, 2002). Milk95 showed>90% denaturation of b-lg and denaturation of a-lactalbumin (a-la)in Milk95 was approximately 3.5 times higher than in Milk72.
Residual PL and PLG derived activities in Milk72 were30.9� 2.3% and 14.0� 2.7%, respectively, whereas no residual PL orPLG derived activity could be detected inMilk95. Denaturation of b-lg and inactivation of PL and PLG are known to be closely correlated(Ismail & Nielsen, 2010; Newstead et al., 2006). The higher heat loadand degree of b-lg denaturation of Milk72 correlates with the lowerresidual PL and PLG derived activity compared to the residual levelsof 50% and 37% PL and PLG derived activity, respectively, found aftersteam injection (150 �C, Dickow, Nielsen, & Hammershøj, 2012).Interestingly, it seems that in direct UHT treatments with ultra-short holding times, PLG appears to have lower heat stabilitythan PL (Table 1). Kinetic studies on the heat inactivation of PL andPLG as well as direct UHT treatments show a similar or higher heatstability of PLG compared with PL at temperatures between 90 and142 �C (Manji & Kakuda, 1986; Rollema & Poll, 1986; Saint-Denis,Humbert, & Gaillard, 2001). This suggests that a different heatinactivation kinetic for PL and PLG applies for direct UHT treat-ments with ultra-short holding times compared with other UHTtreatments and previous kinetic studies.
3.2. Appearance and taste
In Milk72 a bitter flavour was noticeable after 6 weeks thatrapidly intensified, and the milk was extremely bitter after 7 week
of storage. A similar development has been shown for direct UHTtreatments with ultra-short holding time (Huijs et al., 2004). Nobitterness was detected in Milk95.
After 10 weeks storage, a visible difference in the colours be-tween Milks72 and 95 was observed, with Milk72 being less opa-que than Milk95. After 10 weeks, a gel layer was found at thebottom of the bottle of Milk72, which increased in thickness in thefollowing week. Milk95 showed no gelation or sedimentationduring the storage period. The gel was soft and fragile. Along withthis gel layer, floating gel particles appeared in Milk72 after 11weeks storage. After 16 weeks storage, Milk72 was completelygelled and syneresis could be observed. The gel formation andappearance was similar to the description of a plasmin induced gelfound in UHT milk by Kelly and Foley (1997). In contrast to othersimilar studies (Newstead et al., 2006; Topçu et al., 2006), Milk72showed no sedimentation. This difference may arise from theshorter storage time needed for gelation in this study. Milk72already gelled after 80 d of storage, while Newstead et al. (2006)and Topçu et al. (2006) reported gelation after 150e180 d ofstorage.
In addition to the gel formation, a creamy layer appeared on thetop of Milk72 after 80 d of storage, a phenomenon that has beendescribed for milk in connection with plasmin activity byKohlmann et al. (1991). This was most apparent in the samplesadjusted to pH 4.6: due to the homogenisation, small fat globulescould be observed in the supernatant ofMilk95 after centrifugation,while the supernatant of Milk72 was clear with a creamy layer ontop.
3.3. Physical-chemical changes during storage
The pH of Milk72 and Milk95 (Fig. 1) decreased during storage,but the decrease was more pronounced in Milk72, by approxi-mately 0.2 pH-units. The larger decrease in Milk72 can be related tothe residual PL activity in the sample and proteolysis after lysineresidues (Kelly & Foley, 1997; Kohlmann et al., 1991). Interestingly,the pH did not decrease further after the onset of gelation after 10weeks storage.
The colour values of Milk95 only decreased slightly during thestorage period, while the colour values for Milk72 decreasedgradually, becoming less white (decreased L-value), more greenish(higher negative a-value), and less yellow (lower b-value). As canbe seen in Fig. 2, Milk95 showed higher initial a and b colour values,which can be attributed to the higher extent of Maillard reactiondue to the more severe heat treatment (Kneifel, Ulberth, & Schaffer,
Table 1Whey protein denaturation and residual plasmin (PL) and plasminogen (PLG)derived activity in Milk72 and Milk95 after indirect pre-heat treatment and directsteam infusion (>150 �C for >0.2 s).a
Milk Indirect pre-heattreatment
Whey protein denaturation (%) Residual activity (%)
b-lg a-la PL PLG
Milk72 72 �C/180 s 36.7 � 3.2 13.1 � 2.2 30.9 � 2.3 14.0 � 2.7Milk95 95 �C/180 s 94.1 � 1.6 45.5 � 3.3 n.d. n.d.
a The degree of denaturation of whey proteins (b-lg, b-lactoglobulin; a-la, a-lactalbumin) was calculated as the relative difference in the UV peak area of pH 4.6soluble whey proteins before and after heat treatment: results are shown asaverage � standard deviation of three replicates; n.d., non-detectable.
Fig. 1. pH development in milk subjected to direct steam infusion (>150 �C for <0.2 s)and pre-heat treated at 72 �C (�) or 95 �C (,) for 180 s during storage at 20 �C. Resultsare shown as average � standard deviation of three replicates.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 199e207 201
1992). Similar to the pH value, the colour values levelled out withthe onset of gelation, and showed a tendency to increase again forthe a and b colour values. The difference in the L value of Milk72and Milk95 correlates well with the observed visible difference inopacity and could indicate a change in the casein micelles proper-ties. A reduction of the L value has been correlated with an increasein casein micelle size after extensive hydrolysis with PL (Crudden,Afoufa-Bastien, Fox, Brisson, & Kelly, 2005) as well with a dissoci-ation of casein micelles upon tri-sodium citrate addition(O’Sullivan, Kelly, & Fox, 2002). The latter could also be associatedwith PL mediated proteolysis as partially disintegrated casein mi-celles have been found in PL-treated milk (Aroonkamonsri, 1996).
The development in viscosity of Milk72 and 95 is shown in Fig. 2.Viscosity of both milks was constant between 2.0 and 2.5 mPa sduring the first 9 weeks of storage. After 10 weeks storage, theonset of gelation caused the viscosity of Milk72 to increase and itfurther increased rapidly in the following week up to13.6 � 2.6 mPa s. The viscosity of Milk95 did not change during theapplied storage period. A similar observed increase in viscositywith the onset of gelation has been reported previously byKohlmann et al. (1991) and Kelly and Foley (1997).
During the first 10 weeks storage, the particle size distributiondid not change significantly (data not shown). Fig. 3 shows arepresentative volume based particle size distribution of particlesin Milk72 between 0 and 14 weeks storage. Due to the homoge-nisation of the milk, the individual size distributions of casein mi-celles and fat globules could not be distinguished. The 10 week oldmilk shows a mono-modal distribution consisting of casein mi-celles and homogenised fat globules with an average diameter ofapproximately. 190 nm. The change in casein micelles observed inthe colour measurement could not be seen in the particle sizedistribution and is most likely overshadowed by the contribution of
the fat globules to the particle size distribution. After 11 weeksstorage, larger particles between 8 and 10 mm could be observed. Inthe following week, the population of the larger particles increasedwhile the casein micelle/fat globule distribution became narrowerand contributed to a relatively smaller amount of the particles. Thelarge particles were most likely gel fragments. Due to the stirringduring the measurement, the gel particles broke down to smallerfragments resulting in a relatively narrow size distribution. Thebroadening of the distribution towards higher sizes after 14 weeksreflects the increased firmness of the gel.
Fig. 2. Development of Lab colour values and viscosity in milk subjected to direct steam infusion (>150 �C for <0.2 s) and pre-heat treated at 72 �C (�) or 95 �C (,) for 180 s duringstorage at 20 �C. Results are shown as average � standard deviation of three replicates.
Fig. 3. Volume based particle size distribution of milk pre-heat treated at 72 �C for180 s and subjected to direct steam infusion (>150 �C for <0.2 s) after 0 (continuousline), 8 (short-dashed line), 10 (dotted line), 12 (long-dashed line), and 14 (dot-dashline) weeks storage at 20 �C.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 199e207202
The microstructure of the gel found in Milk72 was visualised byconfocal laser scanning microscopy (see Supplementary material).The gel appeared to have a loose, non-homogeneous structure withliquid filled cavities, similar to the microstructure of yoghurts andacid milk gels (Pereira, Matia-Merino, Jones, & Singh, 2006; Torres,Amigo Rubio, & Ipsen, 2012). The micrographs show small fatglobules (0.5e2 mm), that are partially present in clusters andincorporated in the gel structure. The diameter of the fat globules isin linewith the observed particle size distributions. The presence ofagglutinated fat globules could account for the observed creamlayer in Milk72.
3.4. Enzymatic activity and proteolysis during storage
3.4.1. Plasmin and plasminogen derived activityThe development in PL and PLG derived activities in Milk72 are
shown in Fig. 4. As can be seen, PLG derived activity decreasedrapidly, while PL activity increased. This indicates activation of PLGinto PL during storage, and no PLG derived activity was detectableafter 6 weeks storage. A similar development has been reported indirect heated UHT milk (Manji & Kakuda, 1986). The increase in PLactivity could be fitted well to a first order reaction kinetic(k¼0.1828,R2¼0.995),while PLGderived activityon theotherhanddecreased from 0 to 6 week in a linear fashion (y ¼ �104.7x þ 603;R2 ¼ 0.997). The inhibitors of the PL system are heat labile: underpasteurisation conditions (74.5 �C for 15 s) PAI is completely inac-tivated, while about 36% of PI is inactivated (Prado et al., 2006). PAhave been shown to have a similar or even higher heat stability thanPL and PLG (Lu & Nielsen,1993; Saint-Denis et al., 2001), suggestingthat a significant proportion of PA remained inMilk72 after the heattreatment. This, together with the inactivation of PAI, explains thefast rate of PLG activation in Milk72. After 4 weeks storage, the PLactivity inMilk72was comparable to the PL activity found in the lowpasteurised milk (632 � 28 mU mL�1) prior to heat treatment. Thegelation of Milk72 had no apparent effect on PL activity.
The PL and PLG derived activities developed differently and PLactivity continued to increase after depletion of PLG. This devel-opment can be ascribed to other factors affecting PL activity, such asa reduced competitive inhibition of PL by caseins towards thesynthetic substrate in the analysedmilk samples (Bastian, Brown, &Ernstrom, 1991) due to proteolysis.
3.4.2. Changes in protein profiles during storageIn Fig. 5, a comparison of the protein profiles of Milk72 and 95
after 0, 4, 8 and 12 weeks storage is shown. Milk72 showed very
extensive proteolysis during the storage period, while the overallchromatographic pattern for Milk95 remained unchanged. Fromthe UV chromatograms, no statement about the development inaS2-casein could be made due to overlay with upcoming poly-peptides (Fig. 5 AeD). After 4 weeks storage, the b-casein A1 peakdecreased the most, followed by a slight decrease in aS1-casein andb-casein A2. The peak of b-casein A1 disappeared almost completelyafter 8 weeks storage, along with a large decrease in the aS1-caseinpeak. After 12 weeks storage, no intact aS1-casein with 9 phos-phorylations (P) and b-casein A1 was present in the UV chro-matogram (Fig. 5D). Over the storage period, the k-casein peaksseemed to disappear, but developed similarly in both milk types,ruling out proteolysis as a cause for this change in k-casein profile.Instead, the disappearance can be attributed to a broadening of thepeaks due to modifications of the proteins, likely by the Maillardreactions induced by the heat treatment (Gaucher, Mollé, Gagnaire,& Gaucheron, 2008). For Milk95, the same tendency could be seenfor aS2-casein.
Interestingly, significant differences in the peak development ofb-casein A1 and A2 as well as for aS1-casein with 8P and 9P wereobserved. Therefore, the mass spectra of peaks 4e9 were analysedafter 8 weeks storage to investigate the components present in thepeaks. The results are summarised in Table 2 and show that pro-teose peptones 5 and 8 (slow) from b-casein A2 are co-eluting withaS2-casein along with a polypeptide from aS1-casein f(1e124). Thecorresponding polypeptide aS1-casein f(125e199) was co-elutingwith aS1-casein 8P. While the peaks of aS1-casein 9P and b-caseinA1, either were not or only slightly contaminated with poly-peptides, g1- and g2-caseins were co-eluting with b-casein A2 andg3-casein with a-la. This explains the difference in the peakdevelopment of aS1-casein 8 and 9P as well as b-casein A1 and A2. Acomparison of the mass spectra (data not shown) showed that therate of hydrolysis was similar for the two genetic variants A1 and A2
of b-casein and for the differently phosphorylated aS1-caseins. Forthis reason, aS1-casein 9P and b-casein A1 were chosen for a relativequantification of the proteolysis based on the UV chromatograms.
Proteolysis in UHT milk can be caused by PL and heat resistantproteases from, e.g., psychotropic bacteria. The milk used in thisstudy was one day old low-pasteurised milk, which makes it un-likely that the extensive proteolysis in Milk72 was caused by bac-terial proteases. The appearance of proteose peptones and g-caseins suggested that PL was the main actor of proteolysis. Bac-terial proteases are more unspecific in their cleavage sites and arecapable of hydrolysing k-casein (Datta & Deeth, 2001). In Milk72and Milk95 no proteolysis of k-casein was observed, furtherstrengthening the indications that PL was the main active proteasein Milk72.
The hydrolysis of aS1-casein 9P and b-casein A1 in Milk72 duringstorage are shown in Fig 6. Both aS1-casein 9P and b-casein A1
decrease in an inverse sigmoidal manner. Hydrolysis of casein by PLcan usually be described by an exponential decay function(Newstead et al., 2006). In Milk72, a lag phase in the first 2e3weeks storage preceded the exponential decay. This lag phase canbe attributed to the activation of PLG to active PL as shown in Fig. 4.With increasing PL activity, the rate of hydrolysis increased.
The b-casein A1 was hydrolysed faster than aS1-casein 9P, as alsoreported in the literature (Grufferty & Fox, 1988). After 9 weeksstorage, more than 95% of b-casein A1 was hydrolysed, while thesame degree of hydrolysis for aS1-casein 9P was reached after 11week.
The rate of hydrolysis of aS2-casein by PL has been shown to besimilar to b-casein (Grufferty & Fox, 1988). The MS spectrum of theaS2-casein peak at different storage times (data not shown) showedthat aS2-casein was almost completely hydrolysed after 9 weeksstorage, indicating the same rate of hydrolysis as b-casein. Gelation
Fig. 4. Plasmin (�) and plasminogen (6) derived activity in milk pre-heat treated at72 �C for 180 s and subjected to direct steam infusion (>150 �C for <0.2 s) duringstorage at 20 �C. Results are shown as average � standard deviation of three replicates.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 199e207 203
occurred after 95% b-casein A1 and aS1-casein 9P had beenhydrolysed.
The protein composition of the gel formed had the same patternas the liquid (Fig. 7). The relative concentration of residual intact a-and b-caseins and the polypeptides in the gel were higher than inthe liquid, while the concentrations of k-casein and whey proteinswere similar in the gel and in the liquid. As the micrographs
indicated, the gel had a relatively loose structure, which containeda high proportion of liquid phase. This shows that k-casein andwhey proteins were not incorporated into the gel network. To theauthors’ knowledge, the composition of a plasmin induced gel hasnot been reported previously.
Bitterness in Milk72 was noticeable after 6 weeks storage, at atime point when approximately 70% of aS1-casein and approxi-mately 75% of b-casein (and presumably aS2-casein) was hydro-lysed. Bitterness caused by PL has been attributed to peptides from
Fig. 5. Protein profiles of milk subjected to direct steam infusion (>150 �C for <0.2 s) and pre-heat treated at 72 �C (Milk72, solid line) or 95 �C (Milk95, dashed line) for 180 s after0 (A), 4 (B), 8 (C) and 12 (D) weeks storage at 20 �C. The observed proteins at week 0 are k-casein (peaks 1e3), aS2-casein (4), aS1-casein with 8 (5) or 9 (6) phosphorylations, b-casein A1 (7) and A2 (8), a-lactalbumin (9), b-lactoglobulin B (10) and A (11).
Table 2Probable identitya of components in milk pre-heat treated at 72 �C for 180 s andsubjected to direct steam infusion (>150 �C for <0.2 s) after 8 weeks of storage at20 �C from protein profile in peaks 4e9 (see Fig. 5A).
Peak no. Deconvolutedmass (Da)
Theoreticalmass (Da)
Mass error(ppm)
ID
4 8717.61 8717.91 34.9 b-CN A2 f(29e105)12,178.26 12,178.28 2.3 b-CN A2 f(1e105)14,995.20 14,995.15 3.2 aS1-CN 8P f(1e124)25,229.33 25,229.35 5.7 aS2-CN 11P25,309.29 25,309.33 1.0 aS2-CN 12P25,389.32 25,389.31 0.2 aS2-CN 13P
5 8639.36 8639.61 29.0 aS1-CN 8P f(125e199)23,615.68 23,615.75 2.9 aS1-CN 8P
6 23,695.58 23,695.73 6.4 aS1-CN 9P7 24,023.15 24,024.25 4.4 b-CN A1
20,563.65 20,563.88 11.0 b-CN A1 f(29e209)12,679.69 12,680.03 26.3 b-CN f(98e209)
8 11,824.75 11,824.96 20.4 b-CN f(106e209)20,523.57 20,523.86 13.9 b-CN A2 f(29e209)23,984.04 23,984.23 7.8 b-CN A2
9 11,559.45 11,559.64 16.3 b-CN f(108e209)14,187.00 14,187.12 8.3 a-lactalbumin
a Identity is based on mass fingerprinting using average mass and reduced pro-teins and peptides: CN, casein; P, phosphorylated.
Fig. 6. Hydrolysis of aS1-casein 9P (�) and b-casein A1 (6) in milk pre-heat treated at72 �C for 180 s and subjected to direct steam infusion (>150 �C for <0.2 s) duringstorage at 20 �C expressed as the relative change in peak area of UV absorption at214 nm. The arrow indicates the onset of gelation. Results are shown asaverage � standard deviation of three replicates.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 199e207204
b-casein (Harwalkar, Cholette, McKellar, & Emmons, 1993) and aS2-casein (Le Bars & Gripon,1989). The extensive proteolysis necessaryto produce bitterness indicates that either (1) the sensory thresholdof the formed bitter peptides is high or bitterness was masked bythe matrix, or (2) that the cleavage positions that result in bitterpeptides are not preferred by PL and the bitter peptides wereformed after the majority of the preferred cleavage positions hadbeen hydrolysed.
3.4.3. Changes in peptide profiles during storageThe development in the peptide profile of Milk72 (Fig. 8)
showed a rapid increase of peaks with 31e34 min retention timebetween 0 and 4 weeks storage. These peaks were identified asproteose peptones. After 8 weeks storage, more peptides appearedbetween 17 and 30 min. Most of the peptides eluted late in the RP-UHPLC, which has been reported previously to correlate with PLactivity (Datta & Deeth, 2003). The peptide profile changed slightlybetween 12 and 14 weeks storage, although the total peak area ofthe peptide profile of Milk72 remained constant in the same timeperiod. The most profound change in the peptide profile between12 and 14 weeks storage was seen in the proteose peptone peaks.The proteose peptone b-casein A1 f(1e105) eluting at 33.0 mindecreased and b-casein A1 f(29e105) eluting at 32.7 min increased(peptides are indicated by arrows in Fig. 8). This shows that PL wasstill active after gelation of Milk72, as already suggested by the PL
activity measurement. The decreased rate of proteolysis was mostlikely due to a limited accessibility of remaining substrate for PL inthe gel matrix and depletion of preferred cleavage sites.
In Fig. 9, the development in the total peak area of pH 4.6 solublepeptides during storage is shown. The peak area of Milk72 devel-oped in a sigmoidal way and appeared to almost inversely mirrorthe hydrolysis of aS1-casein 9P and b-casein A1 (Fig. 8). After a 2e3week lag phase, the peak area increased rapidly and levelled outafter the onset of gelation. This is in line with the almost completehydrolysis of aS1-casein 9P and b-casein A1 after 11 weeks, showingthat no more material soluble at pH 4.6 was formed. The total peakarea of Milk95 increased only slightly during the storage period,confirming that no significant proteolysis occurred.
3.4.4. Correlation of proteolysis and gel formationProteolysis mediated by PL can cause gelation and increases
transparency of milk and the outcome of proteolysis seem todepend largely on the extent of PL activity and rate of proteolysis inthe milk. Large amounts of added PL have earlier been reported tocause clarification of milk (Crudden et al., 2005), while lowamounts or indigenous residual PL has caused gelation of milk(Kelly & Foley, 1997; Kohlmann et al., 1991; Newstead et al., 2006).
In this study, at the onset of gelation of Milk72 more than 95% ofa- and b-caseins were hydrolysed. Previous studies (Kelly & Foley,1997; Kohlmann et al., 1991; Newstead et al., 2006) showed thatalmost complete proteolysis of b-casein was reached before a gelwas formed, while a-casein on the other hand was either stillpresent to some extent (Kelly & Foley, 1997) or almost completelyhydrolysed (Kohlmann et al., 1991). The low level of b-lg denatur-ation in Milk72, compared to other UHT treatments, and the lowconcentration of b-lg and k-casein in the formed gel suggests thatthe formation and release of a b-lactoglobulin-k-casein complexwas not the main cause of gelation. Most studies on PL in milk havefocussed on b-casein as an indicator of proteolysis. In relation to agegelation, it is important to consider both hydrolysis of aS- and of b-caseins. A part of b-casein has been shown to be highly mobile inthe caseinmicelle (Dalgleish, 2011) and hydrolysis of b-casein couldbe responsible for the reported increase in hydrophobicity(Aroonkamonsri, 1996), while the hydrolysis of aS1-casein could beresponsible for loss of micellar integrity, which in combinationcould lead to the age gelation of milk. Manji and Kakuda (1988)suggested gelation to be a two-stage process, which requiressome degree of proteolysis before time-dependent physical-chemical changes lead to the gelation of milk. This could be a
Fig. 7. Protein profiles of gel particles (solid line) and liquid phase (dashed line) in milkpre-heat treated at 72 �C for 180 s and subjected to direct steam infusion (>150 �C for<0.2 s) after 13 weeks storage at 20 �C.
Fig. 8. Peptide profile of pH 4.6 soluble peptides of milk pre-heat treated at 72 �C for180 s and subjected to direct steam infusion (>150 �C for <0.2 s) at 0, 4, 8, 12 and 14weeks storage at 20 �C. Arrows indicate proteose peptones 8 and 5 of b-casein A1f(29e105) and f(1e105) at 32.7 min and 33.0 min, respectively.
Fig. 9. Development of pH 4.6 soluble peptides of milk subjected to direct steaminfusion (>150 �C for <0.2 s) and pre-heat treated at 72 �C (Milk72, �) or 95 �C(Milk95, ,) for 180 s during storage at 20 �C expressed as total peak area afterintegration of UV absorbance at 214 nm. Results are shown as average � standarddeviation of three replicates.
V.M. Rauh et al. / International Dairy Journal 38 (2014) 199e207 205
reason for the different observations on age gelation of milk causedby PL. If PL activity in the milk is high, the polypeptides could befurther degraded and inhibit the formation of a gel, before physical-chemical changes will take place. In milk with only little residual PLactivity, physical-chemical changes induced by proteolysis couldoccur prior to achieving a degree of proteolysis sufficient to cause agelation, which could be the case for observed sedimentation priorto or instead of gelation (Kelly & Foley,1997; Newstead et al., 2006).This may also explainwhy the degree of proteolysis and the onset ofgelation have not been directly correlated yet.
The results obtained in the present study suggest a gelationmechanism that is similar to acid gelation, with the difference thatproteolysis instead of acid causes a destabilisation of the caseinmicelle. Acidification of milk leads to a reduced repulsion of caseinmicelles due to a reduced negative charge of k-casein, followed by arearrangement of the casein micelles by solubilisation of calciumdue to the pH drop that leads to the formation of a gel network(Lucey & Singh, 2003). While the gelation by plasmin dependsmainly on the rate of hydrolysis, the gel properties of acid gelsdepend largely on the rate of acidification (Anema, 2008). Since thepH in Milk72 is only decreasing slightly, the repulsion by k-casein isstill present in Milk72 and inhibits aggregation of casein micelles,which explains the absence of k-casein in the gel network.
To fully understand the mechanism of PL induced age gelation,these two stages would need to be investigated separately, e.g., byfollowing changes in casein micelle structure in milk with differentdegrees of proteolysis.
4. Conclusions
Our results indicate that PL mediated proteolysis can be a likelycause for age gelation in UHT milk produced with direct steaminfusion (>150 �C,<0.2 s). Activation of PLG to PL could be observedas an increase in PL activity and an increase in the rate of caseinhydrolysis and peptide formation. The observed changes in colourand pH correlated with the proteolysis. The main shelf life limitingfactor for the UHT milk used was the appearance of bitterness,which preceded age gelation. Further studies are necessary toinvestigate the peptide formation by PL and to identify peptidesrelated to the development of bitterness.
Acknowledgements
The Danish Agency for Science, Technology and Innovation(DASTI) is thanked for financial support of the study. Many thanksto Jens Röschmann and Lena Bergström for the help in the ArlaFoods pilot plant and Ulf Andersen, Bettina Mikkelsen (Arla Stra-tegic Innovation Center) and Isabel Celigueta (Nestlé ProductTechnology Center) for the help with the confocal microscopy.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.idairyj.2013.12.007
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Manji, B., & Kakuda, Y. (1986). Effect of storage temperature on age gelation of UHT-milk mrocessed by direct and indirect heating systems. Journal of Dairy Science,69, 2994e3001.
Manji, B., & Kakuda, Y. (1988). The role of protein denaturation, extent of proteol-ysis, and storage temperature on the mechanism of age gelation in a modelsystem. Journal of Dairy Science, 71, 1455e1463.
McMahon, D. J. (1996). Age-gelation of UHT milk: Changes that occur during storage,their effect on shelf life and the mechanism by which age-gelation occurs. Brussels,Belgium: International Dairy Federation.
Newstead, D. F., Paterson, G., Anema, S. G., Coker, C. J., & Wewala, A. R. (2006).Plasmin activity in direct-steam-injection UHT-processed reconstituted milk:effects of preheat treatment. International Dairy Journal, 16, 573e579.
Nicholas, G. D., Auldist, M. J., Molan, P. C., Stelwagen, K., & Prosser, C. G. (2002).Effects of stage of lactation and time of year on plasmin-derived proteolyticactivity in bovine milk in New Zealand. Journal of Dairy Research, 69, 533e540.
O’Sullivan, M. M., Kelly, A. L., & Fox, P. F. (2002). Influence of transglutaminasetreatment on some physico-chemical properties of milk. Journal of DairyResearch, 69, 433e442.
Pereira, R., Matia-Merino, L., Jones, V., & Singh, H. (2006). Influence of fat on theperceived texture of set acid milk gels: a sensory perspective. Food Hydrocol-loids, 20, 305e313.
Prado, B. M., Ismail, B., Ramos, O., & Hayes, K. D. (2007). Thermal stability of plas-minogen activators and plasminogen activation in heated milk. InternationalDairy Journal, 17, 1028e1033.
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Prado, B. M., Sombers, S. E., Ismail, B., & Hayes, K. D. (2006). Effect of heat treatmenton the activity of inhibitors of plasmin and plasminogen activators in milk.International Dairy Journal, 16, 593e599.
Precetti, A. S., Oria, M. P., & Nielsen, S. S. (1997). Presence in bovine milk of twoprotease inhibitors of the plasmin system. Journal of Dairy Science, 80, 1490e1496.
Rauh, V., Bakman, M., Ipsen, R., Paulsson, M., Kelly, A. L., Larsen, L. B., et al. (2013).The determination of plasmin and plasminogen-derived activity in turbidsamples from various dairy products using an optimized spectrophotometricmethod. International Dairy Journal. submitted for publication.
Richardson, B. C. (1983). The proteinases of bovine milk and the effect of pasteur-ization on their activity. New Zealand Journal of Dairy Science and Technology, 18,233e245.
Rollema, H. S., & Poll, J. K. (1986). The alkaline milk proteinase system: kinetics andmechanism of heat-inactivation. Milchwissenschaft, 41, 536e540.
Rollema, H. S., Visser, S., & Poll, J. K. (1983). Spectrophotometric assay of plasminand plasminogen in bovine milk. Milchwissenschaft, 38, 214e217.
Saint-Denis, T., Humbert, G., & Gaillard, J. L. (2001). Heat inactivation of nativeplasmin, plasminogen and plasminogen activators in bovine milk: a revisitedstudy. Lait, 81, 715e729.
Tolkach, A., & Kulozik, U. (2007). Reaction kinetic pathway of reversible and irre-versible thermal denaturation of blactoglobulin. Lait, 87, 301e315.
Topçu, A., Numano�glu, E., & Saldamlı, _I. (2006). Proteolysis and storage stability ofUHT milk produced in Turkey. International Dairy Journal, 16, 633e638.
Torres, I. C., Amigo Rubio, J. M., & Ipsen, R. (2012). Using fractal image analysis tocharacterize microstructure of low-fat stirred yoghurt manufactured withmicroparticulated whey protein. Journal of Food Engineering, 109, 721e729.
Van Asselt, A. J., Sweere, A. P. J., Rollema, H. S., & de Jong, P. (2008). Extreme high-temperature treatment of milk with respect to plasmin inactivation. Interna-tional Dairy Journal, 18, 531e538.
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Supplementary data
S. 1. Microstructure of gel particles in milk pre-heat treated at 72 °C for 180 s and subjected to
direct steam infusion (>150 °C for <0.2 s) after 13 wk of storage at 20 °C. Green areas indicate
protein stained with FITC and red areas fat stained with Nile red (red). The scale is indicated in
the micrographs.
Paper III
Rauh, V. M., Johansen, L. B., Ipsen, R., Paulsson, M., Larsen, L. B., & Hammershøj, M. (2014)
Plasmin Activity in UHT Milk: Relationship between Proteolysis, Age Gelation, and Bitter-
ness
Journal of Agricultural and Food Chemistry, 62, 6852-6860
Errata
First paragraph in materials and methods on page 6853, first column, should say:
Milk Processing. Three batches of 1 day old pasteurized (72 °C for 15 s) and homogenized milk
(1.5% fat) were obtained from Arla Foods Stockholm Dairy (Sweden) during three consecutive
weeks. The milk was preheat treated at 72 °C for 180 s in a tubular heat exchanger before being
subjected to a direct steam infusion heat treatment at >150 °C for <0.2 s (UHT pilot plant,
APV/SPX, Silkeborg, Denmark). After flash cooling to 67 °C, the milk was homogenized (pres-
sure 160/40 bar), further cooled to 20 °C, and packed aseptically in 300 mL glass bottles. The
UHT milk was stored in the dark at 20 °C in a climate chamber for 14 weeks. One bottle was
used once every week of the storage period for analysis. The sterility of each batch of processed
milk was tested according to ISO 483324
.
Plasmin Activity in UHT Milk: Relationship between Proteolysis, AgeGelation, and BitternessValentin M. Rauh,*,†,‡ Lene B. Johansen,† Richard Ipsen,§ Marie Paulsson,∥ Lotte B. Larsen,‡
and Marianne Hammershøj‡
†Arla Foods Strategic Innovation Centre, Rørdrumvej 2, DK-8220 Brabrand, Denmark‡Faculty of Science and Technology, Department of Food Science, Aarhus University, Blichers Alle 20, DK-8830 Tjele, Denmark§Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg, Denmark∥Department of Food Technology, Engineering and Nutrition, Lund University, SE-221 00 Lund, Sweden
*S Supporting Information
ABSTRACT: Plasmin, the major indigenous protease in milk, is linked to quality defects in dairy products. The specificity ofplasmin on caseins has previously been studied using purified caseins and in the indigenous peptide profile of milk. Weinvestigated the specificity and proteolytic pathway of plasmin in directly heated UHT milk (>150 °C for <0.2 s) during 14weeks of storage at 20 °C in relation to age gelation and bitter peptides. Sixty-six peptides from αS- and β-caseins could beattributed to plasmin activity during the storage period, of which 23 were potentially bitter. Plasmin exhibited the highest affinityfor the hydrophilic regions in the caseins that most probably were exposed to the serum phase and the least affinity forhydrophobic or phosphorylated regions. The proteolytic pattern observed suggests that plasmin destabilizes the casein micelle byhydrolyzing casein−casein and casein−calcium phosphate interaction sites, which may subsequently cause age gelation in UHTmilk.
KEYWORDS: age gelation, bitterness, casein, plasmin, proteolysis, UHT milk
■ INTRODUCTION
Proteolysis by plasmin (PL), the major indigenous milkprotease, can greatly affect the quality of dairy products.Proteolysis of caseins by plasmin leads to the formation ofbitter off-flavours in milk and has been linked to the agegelation of UHT milk, making it a critical factor for the shelf lifeof these products.1−3 While PL is an important factor for theripening characteristics of some cheeses,4 PL-induced proteol-ysis can also reduce cheese yield and curd firmness.5,6
PL is the active part of a complex enzyme system in milkconsisting of, apart from PL itself, its zymogen, plasminogen(PLG), which is present in a 6−10 times higher concentrationthan PL, as well as plasminogen activators (PA) and inhibitorsof both PL and PA.7 In contrast to the inhibitors, both PL,PLG, and PA exhibit a high thermal stability.8−10 Heattreatment of milk thus promotes PL-induced proteolysis dueto both reduced inhibitory capacity for residual PL and toincreased activation of PLG into PL. The caseins (CN) in milkare structurally present as colloidal particles (casein micelles),rich in calcium phosphate nanoclusters which are surroundedby the phosphorylated caseins. The casein micelles areinternally stabilized by ionic interactions with calciumphosphate and hydrophobic interactions between caseins,while the protruding κ-casein on the surface sterically stabilizesthe casein micelle externally against aggregation.11 In milk, PLis associated with the casein micelle, ensuring a close proximityto its substrate.12,13 The most susceptible casein for PL in milkis β-CN, giving rise to the well described γ-CN fraction andproteose peptones.14 PL also readily cleaves αS2-CN and to alesser extent αS1-CN, but has little or no activity toward κ-CN
and the major whey protein, β-lactoglobulin (β-lg). Thespecificity of PL toward β-, αS1- and αS2-CN in model studiesusing purified caseins has been described.15−17 The specificityof PL toward caseins in a micellar system has been studied inrelation to the endogenous peptide profile of milk,18,19 but notin relation to extensive proteolysis and quality defects of dairyproducts. The proteolytic specificity of PL could give an insightinto the mobility and interaction of PL with the casein micelles.Age gelation of UHT milk is previously suggested to be
caused by complexation of κ-CN with denatured β-lg duringthe heat treatment of milk. A subsequent release andaggregation of this complex during storage of UHT milkcould then result in gelation.20 Proteolysis is hypothesized tofacilitate the release of the κ-CN−β-lg-complex by hydrolyzingthe caseins that anchor the complex on the surface of the caseinmicelle.21
In a recent study, we investigated the effect of PL activity onphysicochemical changes in milk heated by direct steaminfusion,1 where we found that proteolysis by residual PLresulted in a decrease in pH and whiteness of the milk duringstorage. Simultaneously, PL activity increased during thestorage period, while PLG-derived activity decreased. Thiscorrelated with an increase in the rate of β-, αS1-, and αS2-CNhydrolysis. Bitter off-flavor developed after 7 weeks of storage,and age gelation of the milk was observed after 11 weeks. At
Received: May 5, 2014Revised: June 23, 2014Accepted: June 25, 2014Published: June 25, 2014
Article
pubs.acs.org/JAFC
© 2014 American Chemical Society 6852 dx.doi.org/10.1021/jf502088u | J. Agric. Food Chem. 2014, 62, 6852−6860
this time more than 90% of α- and β-CNs were hydrolyzed.The relatively low degree of β-lg denaturation in the UHT milkof 36.7 ± 3.2% indicated that the age gelation may be caused bya different mechanism than complexation of κ-CN withdenatured β-lg. At this level of β-lg denaturation, only a verylimited fraction of β-lg is associated with κ-CN.22,23
The aim of the present study was to analyze the peptideformation and specificity of PL in this UHT milk and to relatethe peptides found with the previously observed bitter off-flavorand age gelation of the UHT milk during storage.
■ MATERIALS AND METHODSMilk Processing. Three batches of 1 day old pasteurized (72 °C
for 15 s) and homogenized milk (1.5% fat) were obtained from ArlaFoods Stockholm Dairy (Sweden) during three consecutive weeks.The milk was preheat treated at 74 °C for 180 s in a tubular heatexchanger before being subjected to a direct steam infusion heattreatment at >150 °C for <0.2 s (UHT pilot plant, APV/SPX,Silkeborg, Denmark). After flash cooling to 67 °C, the milk washomogenized (pressure 160/40 bar), further cooled to 20 °C, andpacked aseptically in 300 mL glass bottles. The UHT milk was storedin the dark at 20 °C in a climate chamber for 14 weeks. One bottle wasused once every week of the storage period for analysis. The sterility ofeach batch of processed milk was tested according to ISO 4833.24
Sample Preparation. A volume of 15 mL of milk was taken fromeach bottle and adjusted to pH 4.6 with 1 M HCl followed bycentrifugation at 11000g for 10 min at 4 °C. The clear supernatant wasrecovered and stored at −20 °C until further analysis. At the time ofanalysis, the samples were thawed for 60 min at room temperature.One mL of the sample was centrifuged at 13200g at 5 °C for 10 minand used for the analysis. Sample preparation was performed induplicate.Peptide Analysis. Ultra-high-performance liquid chromatography
(uHPLC) was performed on an Agilent 1290 Infinity uHPLC (AgilentTechnologies, Santa Clara, CA, USA) coupled to a quadrupole-time-
of-flight mass spectrometer (Q-TOF MS, Agilent 6530 Accurate MassQ-TOF LC/MS, Agilent Technologies) with a Jetstream interface.
Depending on the peptide concentration, 10 or 20 μL of the samplewas injected to the uHPLC-Q-TOF-MS system. The column was anXbridge BEH 300 (rp-C18, 3.5 μm 2.1 mm × 250 mm) (Waters,Milford, MA, USA) operated at 45 °C. Buffer A was Milli-Q water with0.05% trifluoroacetic acid (TFA) (v/v and buffer B acetonitrile with0.1% (v/v) TFA. A linear gradient was applied with 100% buffer Afrom 3 min, 100% to 45% buffer A at 45 min at a flow rate of 0.3 mLmin−1. Peptides were monitored at 214 nm.
The Q-TOF MS was run in 4 GHz high resolution mode. TheJetstream interface added a sheath gas at 350 °C with a flow of 8 Lmin−1. A nozzle voltage of 100 V was applied to improve theionization. The drying gas temperature was set to 325 °C at a flow rateof 10 L min−1. The capillary voltage was set to 2.5 kV in the first timesegment (3.0−24.0 min) and to 4.0 kV in the second time segment(24.0−35.2 min) to achieve optimal ionization for both small and largepeptides. Spectra in the mass range from 300 to 3200 m/z wererecorded in positive mode with a resolution of 20000 in the Q-TOF.Tandem mass spectra of the two most abundant ions with a chargestate of 1, 2, or 3 in each MS spectrum were obtained in collision-induced dissociation (CID) mode (collision gas nitrogen, isolationwidth 1.3 m/z (narrow) at 5 different fixed collision energies andflexible collision energies optimized for larger peptides. The fixedcollision energies were 10, 20, 30, 50, and 100 V, and the flexibleenergy settings were based on charge state and m/z (singly charged:offset 18 V, slope 3 V m/z−1; doubly charged: offset 2 V, slope 2.2 Vm/z−1; triply charged: offset 3.5 V, slope 1.5 V m/z−1). Mass spectraand UV chromatograms were analyzed by MassHunter software(version B 6.01, Agilent Technologies).The MS spectra wereinvestigated for potential peptides formed by plasmin, which werenot identified by MS/MS, using the “Find by Formula” feature inMassHunter. Matching ions and isotopic patterns were furtherevaluated with regard to their retention time.
Database Search. The obtained tandem mass spectra wereanalyzed with Mascot v2.4 (Matrix Science, London, UK). Tandemmass spectra were searched against a custom database containing
Table 1. Presence and Identity of pH 4.6 Soluble Peptides from αS1-CN Formed by Plasmin in Directly Heated UHT Milk;Peptides with Q Values >1400 Are Potentially Bitter and Are Shown in Bold
storage time
peptidea mass day 1 4 weeks 8 weeks 14 weeks Q valueb
αS1-CN f(1−7) 874.55 x x x 1777αS1-CN f(1−22) 2615.48 x x x 1297αS1-CN f(23−34) 1383.72 x x x 1833αS1-CN f(23−36) 1640.86 x x 1718αS1-CN f(35−42) 945.51 x x 1030αS1-CN f(80−90) 1336.67 x x x x 1054αS1-CN f(80−100) 2585.37 x 1342αS1-CN f(80−102) 2826.54 x 1396αS1-CN f(91−100) 1266.70 x x 1660αS1-CN f(91−102) 1507.88 x x 1710αS1-CN f(91−103) 1635.97 x x 1694αS1-CN f(91−105) 1927.14 x 1759αS1-CN f(100−105) 678.45 x x 1753αS1-CN f(101−124) + P 2916.53 x 1358αS1-CN f(103−124) + P 2675.35 x x x 1304αS1-CN f(104−119) + P 1950.95 x 1339αS1-CN f(104−124) + P 2547.26 x x x 1294αS1-CN f(106−119) + P 1659.79 x x 1218αS1-CN f(106−124) + P 2256.10 x x x 1201αS1-CN f(120−124) 614.32 x x 1152αS1-CN f(125−132) 909.47 x 756αS1-CN f(194−199) 747.36 x x x 1703
aPhosphorylations are indicated after the peptide. bQ value was calculated according to Ney (1971) in cal mol−1. Presence of peptides at differentstorage times is indicated by ‘x’.
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known genetic and PTM (post translation modification) variants ofmajor milk proteins. The following search parameters were applied:protease: unspecific; mass tolerance for precursor ions 15 ppm and 0.6Da for product ions; variable modifications: phosphorylations serine,oxidation methionine. Only identified peptides (expect score <0.05)that were present in at least two of the three replicates wereconsidered.Relative Quantification of Identified Peptides. Peaks in the
UV chromatogram were identified by their corresponding MS spectraand retention time of the identified peptides by MS/MS. Theretention time shift between the UV and MS signal was approximately0.25 min. Only peaks without or with only minor overlaps with othercomponents in the MS spectra were considered. In order to comparethe rate of peptide formation, the integrated UV peak area was dividedby a calculated molar extinction coefficient (ε) for the peptide asdescribed previously.25 Phosphorylation of serine was not taken intoaccount. Peak areas were further normalized for the initial caseindistribution to correct for the differences in peptide concentration.The casein composition was based on the UV peak area corrected forthe calculated ε (αS1-CN: 30%, αS2-CN: 13%, β-CN: 44%, κ-CN:13%). Calculated ε in M−1 cm−1 were 346843 (αS1-CN), 375032 (αS2-CN), 331417 (β-CN) and 279859 (κ-CN).Assessment of Bitterness. Bitterness of peptides was evaluated
by applying Ney’s Q-rule based on peptide hydrophobicity.26 Anaverage hydrophobicity Q of the peptide is calculated as the sum of theamino acids side chains hydrophobicity divided by the number ofamino acid residues of the peptide. Peptides with a Q value >1400 cal/res−1 are considered bitter
■ RESULTS AND DISCUSSION
A total of 84 different casein-derived peptides were identifiedby the uHPLC-Q-TOF analysis (Tables 1−4). In the 1 day oldmilk (analyzed 1 day after processing), only 11 peptides couldbe identified by MS/MS. This number is small compared toother recent studies, which used more profound peptideextraction and concentration methods.18,19 The main objectivein this study, however, was to study the proteolysis anddevelopment of peptides during storage rather than the peptideprofile of the milk used for the UHT treatment. The majorityby number of the peptides identified were derived from αS2-CN(36 peptides), followed by αS1-CN (31) and β-CN (17).During the entire storage period, no peptides derived from κ-CN were detected, indicating that indigenous proteases otherthan PL, such as cathepsin D, were probably heat inactivated ascould be expected due to their lower heat stability comparedwith PL and also that the raw material had not contained heatstable proteases from psychotrophic bacteria, which are knownto be able to hydrolyze κ-CN.27,28 More than 75% of thepeptides (66 peptides out of 79) could be assigned to be theresult of PL action solely (Table 4). In the remaining 18identified peptides, PL was responsible for either the N- or C-terminal cleavage in 11 of the 18 identified peptides.Identified Peptides Formed by Plasmin. Identified
peptides derived from αS1-CN and the storage time of theirappearance is shown in Table 1. The position of the peptideswithin in the αS1-CN sequence is shown in Figure 1. The onlypeptide present after 1 day of storage was αS1-CN f(80−90),suggesting Lys79-His80 and Arg90-Tyr91 to be the initial sitescleaved by PL. After 4 weeks of storage, the peptides identifiedoriginated from the N-terminal region of αS1-CN (Arg22-Phe23and Lys7-His8), from the central, lysine-rich region (Lys102-Lys103, Lys103-Tyr104, Lys105-Val106, and Lys124-Glu125) as well asthe C-terminal end with cleavage at Lys193-Thr194. The peptidesαS1-CN f(91−102), αS1-CN f(91−103), and αS1-CN f(91−105)were not identified until after 8 or 14 weeks of storage,
indicating that Lys79-His80 and Lys124-Glu125 are the two majorcleavage sites in the central region of αS1-CN. The peptides αS1-CN f(23−34), αS1-CN f(23−36), and αS1-CN f(35−42)appeared after 4−8 weeks of storage. This implies a sequentialcleavage of the N-terminal region of αS1-CN by PL beginning atArg22-Phe23 and subsequently Lys34-Glu35 and Lys36-Val37.Peptides containing these cleavage sites Arg100-Leu101, Arg119-Leu120, and Lys132-Glu133 occur only after 8 or 14 weeks ofstorage and seem not to be preferred by PL. The late or slowcleavage at these positions is most likely due to the preferenceof PL for cleaving after lysine rather than after arginine29 andtheir close proximity to the major cleavage sites makes a furtherhydrolysis in the vicinity unlikely.The identified cleavage sites of PL and the order of
occurrence are in good agreement with previous studies usingpurified αS1-CN
17,29 with the exception of the cleavage sites atArg151-Gln152, a proposed major cleavage site by McSweeney etal.,17 and at Lys58-Gln59. Relevant peptides and polypeptidescontaining the cleavage site Arg151-Gln152 (αS1-CN f(125−151),αS1-CN f(133−151), αS1-CN f(106−151), and αS1-CN f(152−199)) were not detected. The cleavage site Arg151-Gln152 islocated in middle of the most hydrophobic region of αS1-CN(Figure 4A), which has been identified as the main polymer-ization and interaction region of αS1-CN via hydrophobicinteractions.30 In contrast to purified αS1-CN, this region islikely to be inaccessible for PL and Arg151-Gln152 and was alsonot hydrolyzed by trypsin in a micellar system.31,32 The secondnot-present cleavage site, Lys58-Gln59, is placed between thetwo phosphoserine clusters of αS1-CN (residues 41−51 and61−70). The phosphoserine residues tightly bind to thecalcium phosphate clusters in the casein micelle, which makesthis region inaccessible for enzymatic hydrolysis.31,33
The largest numbers of peptides present as a result of PLactivity derives from αS2-CN (Table 2). After 1 day of storage,peptides originating from the N- and C-terminal region of αS2-CN were present (Figure 2). The C-terminal region of αS2-CNhas previously been shown to be exposed and easily accessiblefor hydrolysis,34 and the appearance of αS2-CN f(182−188) andαS2-CN f(174−181) after 4 weeks of storage suggests asequential hydrolysis of the C-terminal region. Initial cleavagesites are Lys197-Thr198 and Lys188-Ala189, followed by Lys181-Thr182 and Lys173-Phe174. After the same storage time, peptidesoriginating from the central region of αS2-CN were formed byhydrolysis of Arg114-Asn115, Lys136-Lys137, Lys137-Thr138, Lys149-
Figure 1. Primary structure of bovine αS1-CN B-8P. Phosphoserineresidues are indicated by circles. Open circle indicates a possiblephosphorylation at Ser41. Identified pH 4.6 soluble peptides formed byplasmin during storage at 20 °C are indicated by arrows.
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Lys150, and Lys150-Thr151. Earlier studies suggest that thecleavage sites Lys149-Lys150 and Lys150-Thr151 are preferred overArg114-Asn115,
16 but the simultaneous appearance of αS2-CNf(115−136), αS2-CN f(137−149), and αS2-CN f(138−150)indicate that PL readily hydrolyzed all of these cleavage sites.After 8 weeks of storage peptides originating from the region151−173 in αS2-CN were identified. The late appearance ofthese peptides indicates a sequential hydrolysis of this regionfrom an N- and C-terminal direction after the preferredcleavage sites Lys150-Thr151 and Lys173-Phe174 are hydrolyzed.The least preferred cleavage sites in αS2-CN seem to be Arg125-Glu126 and Lys152-Leu153, which are either in close proximity tophosphoserine residues or to major cleavage sites (Figure 2).Although the region 25−114 contains eight potential cleavagesites for PL, only αS2-CN f(71−80) and αS2-CN f(25−45)could be identified. Masses corresponding to peptides includingthe cleavage site Lys91-Phe92, Lys32-Glu33, and Lys41-Glu42 werenot present in the milk. The not-present cleavage at Lys91-Phe92is located in a hydrophobic region of αS2-CN (Figure 4B),which may be inaccessible for enzymatic hydrolysis.35 Cleavageof this bond can be additionally hindered by Pro93 whichinduces a change in the secondary structure of αS2-CN.
35 Lys32-Glu33 and Lys41-Glu42 are located in the proximity of the two
Table 2. Presence and Identity of pH 4.6 Soluble Peptides from αS2-CN Formed by Plasmin in Directly Heated UHT Milk;Peptides with Q Values >1400 Are Potentially Bitter and Are Shown in Bold
storage time
peptidea mass day 1 4 weeks 8 weeks 14 weeks Q valueb
αS2-CN f(1−21) + 4P 2745.99 x x x x 880αS2-CNf(1−24)c + 4P 3131.19 x x x x 851αS2-CN f(25−45)d 2380.15 x 1019αS2-CN f(71−80) 1245.64 x x 1245αS2-CN f(115−125) 1194.67 x 1331αS2-CN f(115−136) + 2P 2587.16 x x x 939αS2-CN f(115−137) + 2 P 2715.25 x x 963αS2-CN f(115−149) + 3 P 4162.85 x x 971αS2-CN f(115−150) + 3 Pc 4290.94 x x x 986αS2-CN f(137−149) + P 1593.70 x x x 1025αS2-CN f(137−150) + P 1721.80 x x x 1059αS2-CNf(138−149) + P 1465.61 x x x 986αS2-CN f(138−150) + P 1593.70 x x x 1025αS2-CN f(150−165) 1990.12 x x 1215αS2-CN f(151−165) 1862.03 x x 1177αS2-CN f(151−166) 1990.12 x x 1197αS2-CN f(151−173) 2893.61 x x 1177αS2-CN f(153−165) 2632.88 x 1208αS2-CN f(166−173) 1049.60 x x 1176αS2-CN f(167−173) 921.50 x x 1130αS2-CN f(171−181) 1397.77 x x 1762αS2-CN f(174−181) 978.55 x x x 1846αS2-CN f(174−188) 1863.00 x x 1463αS2-CN f(182−188) 902.46 x x x 971αS2-CN f(182−197) 1982.06 x x x 1434αS2-CN f(182−207) 3214.78 x x 1644αS2-CN f(189−197) 1097.61 x x x 1793αS2-CN f(189−199) 1326.75 x x 1644αS2-CNf(189−207) 2330.33 x x x x 1892αS2-CNf(198−205) 974.59 x x 1814αS2-CNf(198−207) 1250.74 x x x x 1980αS2-CNf(200−207) 1021.60 x x x 2233
aPhosphorylations are indicated after the peptide. bQ value was calculated according to Ney (1971) in cal mol−1. cPeptide was identified by MS only.dIntramolecular disulfide bond. Presence of peptides at different storage times is indicated by ‘x’.
Figure 2. Primary structure of bovine αS2-CN A-11P. Phosphoserineresidues are indicated by circles. The dotted line between Cys36 andCys40 indicates the intramolecular disulfide bridge. Identified pH 4.6soluble peptides formed by plasmin during storage at 20 °C areindicated by arrows.
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cysteine residues of αS2-CN. The majority of the αS2-CNmolecules is present in a form with an intramolecular disulfidebond, which induces a tight loop in the protein34,36 which maylimit the accessibility of Lys41-Glu42 and Lys32-Glu33 for PL. Toour knowledge, the cleavage of Arg45-Asn46, Lys70-Ile71, Lys80-Ile81, Arg125-Glu126, Lys166-Ile167, and Arg205-Tyr206 has not beenreported earlier for PL in either model studies or as part of theindigenous peptide profile in milk. This could be attributed tothe low concentration of αS2-CN in milk or a differentconformation of αS2-CN in model systems.31,35
In comparison to αS2-CN, only a relatively small number ofpeptides was identified derived from β-CN (Table 3). Thepeptides β-CN f(1−28), β-CN f(1−29), β-CN f(29−48), andβ-CN f(98−107) were present in the milk after 1 day ofstorage. After 4 weeks of storage, all the observed peptidesexcept β-CN f(33−48) and β-CN f(1−25) were present. Theidentified peptides mainly derived from cleavage around thetwo major cleavage sites of PL in β-CN, Lys105-His106, andLys107-Glu108 (Figure 3). The peptide β-CN f(170−176) wasthe only identified peptide originating from the hydrophobic C-terminal part of β-CN and in contrast to earlier studies usingtrypsin,31,37 no peptides were identified from hydrolysis ofArg183-Asp184 and Arg202-Gly203.
Peptides Formed by Other Proteases. Peptides thatwere identified in the UHT milk samples and which cannot beassigned to be a result of PL activity alone are summarized inTable 4. Most peptides deriving from αS1-CN or β-CN indicateactivity of cathepsins alone or in combination with PL.28,38,39
Peptides formed by cathepsins alone were identified after 1 dayof storage or after 4 weeks of storage in the case of αS1-CNf(1−14). Peptides formed by the combined activity ofcathepsins and PL on the other hand appear after 4−8 weeksof storage. This indicates that cathepsins were active in the rawor pasteurized milk, but inactivated by the UHT treatment andthat the polypeptides formed by cathepsins were furtherhydrolyzed by residual PL to smaller peptides. With theexception of cathepsin D, the heat stability of cathepsins islargely unknown. Although cathepsin D in its active form canpartially survive HTST pasteurization,40 it is unlikely thatsufficient active cathepsins are present after the UHT treatmentto significantly contribute to the observed proteolysis. Thepeptides deriving from αS2-CN originate from the C-terminal ofthe protein. With the exception of αS2-CN f(201−206), thepeptides contained the PL cleavage site Lys197-Thr198. Theapparently unspecific C-terminal cleavage site of the peptidessuggests activity of carboxypeptidases in the raw milk or heat-induced hydrolysis.41,42
Peptide Development during Storage. The presence ofidentified peptides after different storage periods gives a goodindication of preferred and uncleaved cleavage sites of PL in theUHT milk system, but only gives limited information on theactual concentration and development of the peptides.Semiquantitative analysis of the peptides using MS spectrawas not possible because their response factors are unknownand their intensity might be affected by ion suppression ofcoeluting components. The UV chromatograms at 214 nmwere therefore used for a relative quantification and comparisonof the peptides. Due to substantial overlap of large areas in thechromatogram, only a limited number of peaks could be usedfor such relative quantification. Quantified peptides includedαS1-CN f(1−22), αS1-CN f(106−124), αS2-CN f(1−21), αS2-CN f(1−24), αS2-CN f(115−150), as well as β-CN f(1−28)and β-CN f(29−48). A comparison of the UV peaks and theextracted ion chromatograms are shown in the SupportingInformation together with MS spectra of the peaks.
Table 3. Presence and Identity of pH 4.6 Soluble Peptides from β-CN Formed by Plasmin in Directly Heated UHT Milk;Peptides with Q Values >1400 Are Potentially Bitter and Are Shown in Bold
storage time
peptidea mass day 1 4 weeks 8 weeks 14 weeks Q valueb
β-CN f(1−25) + 4P 3121.26 x x 1006β-CN f(1−28) + 4P 3476.48 x x x x 1057β-CN f(1−29) + 4P 3604.58 x x x x 1072β-CN f(29−48) + P 2559.13 x x x x 818β-CN f(33−48) + P 2060.82 x x 614β-CN f(98−105) 872.48 x x x 1328β-CN f(98−107) 1137.63 x x x x 1262β-CN f(100−105) 645.32 x x x 1238β-CN f(100−107) 910.47 x x x 1179β-CN f(106−113) 1012.52 x x x 1655β-CN f(108−113) 747.36 x x x 1873β-CN f(170−176) 779.49 x x x 1777
aPhosphorylations are indicated after the peptide. bQ value was calculated according to Ney (1971) in cal mol−1. Presence of peptides at differentstorage times is indicated by ‘x’.
Figure 3. Primary structure of bovine β-CN A2-5P. Phosphoserineresidues are indicated by circles. Identified pH 4.6 soluble peptidesformed by plasmin during storage at 20 °C are indicated by arrows.
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A comparison of peptides originating from the N-terminus oftheir parent proteins is shown in Figure 5. The peptide β-CNf(1−28) had the highest formation rate. During the first 4−5weeks of storage, the relative concentration increasedexponentially, reflecting the previously described increase inPL activity and rate of peptide formation. From 5 weeks ofstorage and further on, the relative concentration of β-CN f(1−28) increased almost linearly and then leveled out after 12weeks of storage. The decrease in the rate of formation couldbe linked to changes in the matrix induced by the startinggelation of the milk after 11 weeks of storage. Furtherdegradation of β-CN f(1−28) to β-CN f(1−25) may alsoreduce the concentration. Since the proteose peptones β-CNf(1−105) and β-CN f(1−107) were still present after 14 weeksof storage (data not shown), substrate depletion can beexcluded as a cause for the reduced rate of formation. Thepeptide αS2-CN f(1−24) showed the same initial rate offormation as β-CN f(1−28) and leveled off after 6 weeks ofstorage. The peptide αS2-CN f(1−21) exhibited a lower rate offormation than αS2-CN f(1−24), but in contrast to αS2-CNf(1−24), continued to increase after 6 weeks of storage. Thisindicates a hydrolysis of αS2-CN f(1−24) to αS2-CN f(1−21)that may be caused by substrate depletion of the cleavage siteLys24-Asn25. The development of αS1-CN f(1−22) differedfrom the other N-terminal peptides. The initial lag phase wasnot observable for αS1-CN f(1−22), and the overallconcentration was significantly lower compared to that of theN-terminal peptides derived from αS2- and β-CN. The cleavagesites for these peptides in αS2- and β-CN are in very hydrophilicdomains of the proteins (Figure 4 A−C), while the cleavage siteArg22-Phe23 in αS1-CN is in a hydrophobic domain involved incasein−casein interactions.43
Peptides being present as the result of two proteolyticcleavages are shown in Figure 6. They exhibited an extended lagphase compared to the N-terminal peptides, due to the timeneeded to form their precursors. As discussed above, αS2-CN
f(115−150) originates from two of the most preferred cleavagesites in αS2-CN and showed the highest formation rate. Thepeptide αS1-CN f(106−124) developed almost in the samemanner as αS1-CN f(1−22) within the first 6 weeks of storagebut showed a higher rate of formation from 7 weeks of storageon. These findings further highlight the preference of PL forcleavage sites located in the hydrophilic regions of the caseins.Gagnaire et al. (1998) found that the proteose peptones β-CNf(1−105/7) were retained in the casein micelle, while β-CNf(29−105/7) was released into the serum phase.31 This releaselimits further hydrolysis of these proteose peptones by thecasein-associated PL, which explains why β-CN f(29−48) wasonly formed in very small amounts.
Destabilization of Casein Micelles by Proteolysis. PLenters the milk from the blood44 and associates itself with thecasein micelle.45 Whether PL is able to enter the micellarstructure or is located entirely on the surface of the caseinmicelle is unknown. It becomes obvious from Figure 3 that themajority of the major cleavage sites of PL within the caseins,similar to those of trypsin,31,32 are located at the mosthydrophilic and serum-exposed regions of the proteins. β-CN ismainly present in the interior of the casein micelle46 and isproposed to stabilize water or serum channels within the caseinmicelle.47,48 αS2-CN is involved in the binding to calciumphosphate clusters, regions that are hypothesized to besurrounded by hydrophobic domains11 and likely to beinaccessible for PL. The rapid cleavage, within 4 weeks ofstorage, of the N-terminal regions of β-CN and αS2-CN impliesthat PL is able to penetrate the casein micelle. In relation to adestabilization of the casein micelle by proteolysis, it is notablethat the minor cleavage sites, together with some of the majorcleavage sites, are located at the boundary between hydrophilic,hydrophobic, or phosphorylated regions of the caseins (Figure4A−C). Thus, PL hydrolyses around the regions essential forthe internal integrity and stabilization of the casein micelle, i.e.hydrophobic interaction between caseins, interactions of
Table 4. Presence and Identity of pH 4.6 Soluble Casein Derived Peptides Formed by Other Proteases in Directly Heated UHTMilk; Peptides with Q Values >1400 Are Potentially Bitter and Are Shown in Bold
storage time proteaseb
peptide mass day 1 4 weeks 8 weeks 14 weeks Q valuea N-terminal C-terminal
αS1-CN f(1−8) 1011.62 x x x 1618 − ?αS1-CN f(1−14) 1663.93 x x x 1309 − CBαS1-CN f(1−21) 2459.38 x x x x 1324 − CGαS1-CN f(1−24) 2909.61 x x x 1410 − CB,CDαS1-CN f(3−7) 621.40 x 1598 CB PLαS1-CN f(24−36) 1089.59 x x 1646 CB,CD PLαS1-CN f(25−36) 1236.66 x x 1563 CB,CD PLαS1-CN f(180−199) 2215.05 x x x x 1232 CG −αS1-CN f(185−199) 1688.81 x x x 1232 ? −αS2-CN f(198−202) 556.36 x x x 1844 PL CPαS2-CN f(198−203) 719.42 x x x 2015 PL CPαS2-CN f(198−204) 818.49 x x 1969 PL CPαS2-CN f(201−206) 809.44 x 2292 ? CPβ-CN f(96−105) 1087.61 x x x 1180 CD,CG PLβ-CN f(96−107) 1352.76 x x 1262 CD,CG PLβ-CN f(106−125) 2390.16 x x 1482 PL CDβ-CN f(108−125) 2125.00 x x 1535 PL CDβ-CN f(163−169) 761.43 x 909 CB,CD PL
aQ value was calculated according to Ney (1971) in cal mol−1. bPossible responsible proteases for N- and C-terminal cleavage are indicated: CB,cathepsin B; CD, cathepsin D; CG, cathepsin G; PL, plasmin; CP, carboxypeptidase; −, no cleavage; ?, unknown protease. Presence of peptides atdifferent storage times is indicated by ‘x’.
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caseins with calcium phosphate, and ionic or salt interactionsbetween caseins. Cleavage of these regions in the casein micellewould lead to a loosening of the micelle structure, whichcorrelates with the reported increase in micelle size afterincubation with PL.49 A looser structure is hypothesized toallow PL to reach previously inaccessible regions, which canexplain the initial resistance of αS1-CN to hydrolysis.Age gelation of UHT milk has been proposed to be a two-
stage process of proteolysis and physicochemical changes thatlead to the gelation.50 Proteolysis-induced age gelation of UHTmilk by PL which does not involve the κ-CN−β-lg -complex,could occur after a critical number of the casein−casein andcasein−calcium phosphate interaction sites have been hydro-lyzed and the remaining interactions are not sufficient tomaintain a micellar arrangement. While at the point of gelation,more than 95% of the αS- and β-CN were hydrolyzed,1 thepeptide development showed that far from all cleavage sites
were hydrolyzed completely. This indicates that a large numberof amphiphilic and charged polypeptides were present in themilk system that could form and stabilize a gel network. Whilesome proteolysis is needed for gelation, the extent ofproteolysis has not yet been related with gelation. The lackof consistency in degree of proteolysis in relation to agegelation and PL activity described in previous studies could alsobe attributed to differences in processing conditions and levelsof PL activity (direct or indirect UHT treatments, addition ofPL to UHT milk).2,50−53 Different processing conditions cangreatly affect the protein matrix in milk, such as the degree ofwhey protein denaturation on casein micelles,22 therebyinfluencing the substrate accessibility and the proteolyticpattern of PL in milk. A high PL activity could result in afast breakdown of polypeptides to small peptides, which areunable to form a gel. Our results suggest that the proteolytic
Figure 4. Hydrophobicity distribution in αS1-CN (A), αS2-CN (B),and β-CN (C) with major (●) and minor (○) cleavage sites ofplasmin and phosphoserine clusters (*). Classification of major andminor cleavage sites is based on Tables 1−3. Hydrophobicity wascalculated according to Tanford (1962) using an average of sevenamino acids with a relative weight for the amino acid center being oneand 0.75, 0.5, and 0.25 for the amino acids one, two, or three positionsfrom the center amino acid. Positive values indicate hydrophobic andnegative values, hydrophilic regions.
Figure 5. Development of casein-derived N-terminal peptides formedby plasmin in milk preheat treated at 72 °C for 180 s and subjected todirect steam infusion (>150 °C for <0.2 s) during storage at 20 °C.Relative concentration is based on peak area of UV absorbance at 214nm corrected for the peptides theoretical molar extinction at 214 nm.Peptide concentration is normalized for αS2-CN.
Figure 6. Development of casein-derived peptides formed by plasminin milk preheat treated at 72 °C for 180 s and subjected to direct steaminfusion (>150 °C for <0.2 s) during storage at 20 °C. Relativeconcentration is based on peak area of UV absorbance at 214 nmcorrected for the peptides theoretical molar extinction at 214 nm.Peptide concentration is normalized for αS2-CN.
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pathway of caseins by PL is more important for thedestabilization of the casein micelles than the overall decreasein the level of intact caseins.Potential Bitter Peptides. Potential bitter peptides with a
Q value >1400 cal mol−1 are highlighted in Table 1−4.Bitterness of peptides from β-CN was found in the γ-CNfraction after hydrolysis with PL,3 and analysis of tryptic digestsof β-CN have shown that β-CN f(203−209) is extremelybitter.37 The potential bitter peptides found in the presentstudy were β-CN f(108−113) and β-CN f(170−176), whichhave previously been described sensorially as tickling andburning.37 The bitter peptide β-CN f(203−209) was notidentified in the UHT milk or was only present in a very lowconcentration. Previously described bitter peptides derivingfrom αS1-CN, that can be formed by PL, are αS1-CN f(23−34)and αS1-CN f(91−100).54,55 Besides αS1-CN f(91−100), allpeptides from residue 91 to 100−105 found in the milk had Qvalues >1400, and were potentially bitter. The smaller peptidesαS1-CN f(1−7) and αS1-CN f(194−199) were highly hydro-phobic, and could have contributed to bitterness. The peptidesαS2-CN f(182−207), αS2-CN f(189−207), and αS2-CN f(198−207) have been correlated with bitterness and were also presentearly in the storage period in the UHT milk.16 Besides αS2-CNf(182−188), all peptides deriving from the C-terminal region ofαS2-CN from residue 171 and onward are potentially bitter.Cleavage of Arg205-Tyr206 in αS2-CN gives rise to the bitterdipeptide Tyr-Leu.56 The identified potential bitter peptidesindicate that the bitterness caused by PL might mainly becaused by hydrolysis of αS1- and αS2-CN, rather than β-CN aspreviously reported.3 Several peptides formed by the combinedaction of PL and cathepsins were potentially bitter as well. Mostof these peptides derive from the N-terminal region of αS1-CNand the C-terminal region of αS2-CN.A bitter off-flavor was detected in the milk after 6 weeks of
storage, which intensified rapidly within 1 week of storage.1
The concentration and sensory thresholds of the potentialbitter peptides in the UHT milk are unknown, and thebitterness cannot be attributed to a specific peptide. We suggestthat either the overall concentration of bitter peptides reachedthe sensory threshold after 7 weeks of storage or that one orseveral bitter peptides with a lower sensory threshold wereformed very rapidly in the storage period between 5 and 7weeks. Similar to trypsin, PL has the capability to form a rangeof potential bitter peptides, but further studies are necessary toconfirm the bitterness of these peptides and their sensorythreshold.In conclusion, the special UHT process used allowed the
investigation of specificity and effect of PL in a sterile milksystem, without causing substantial changes to the milk by theheat treatment. PL showed a different specificity in the milksystem compared to previous model studies, and 6 newcleavage sites of PL in αS2-CN could be identified. The relativequantification of peptides and time of their appearance alloweda direct comparison of formation rates of peptides to assess theaffinity of plasmin to certain cleavage sites. We could show thatPL is able to produce potentially bitter peptides and bitternesswas most likely caused by peptides arising from αS1- and αS2-CN. The observed proteolytic pattern indicated that PL-mediated proteolysis can destabilize the casein micelle andexplain proteolysis-induced age gelation of milk.
■ ASSOCIATED CONTENT*S Supporting InformationSpectra of peptides used for relative quantification and proteinsin Mascot database used for identification. This material isavailable free of charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATIONCorresponding Author*(V.M.R.) Phone: +45 87 33 2783. Fax: +45 87 46 6688. E-mail: [email protected] Danish Agency for Science, Technology and Innovation(DASTI) is thanked for financial support of the study.NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSWe thank laboratory technicians Vibeke Mortensen and BetinaHansen at the Arla Strategic Innovation Center for assistancewith the analysis and helpful discussions.
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bridges in monomers of κ- and αs2-casein from bovine milk. J. DairyRes. 1994, 61, 485−493.(37) Bumberger, E.; Belitz, H. D. Bitter taste of enzymic hydrolysatesof casein. I. Isolation, structural and sensorial analysis of peptides fromtryptic hydrolysates of beta-casein. Z. Lebensm. Unters. Forsch. 1993,197, 14−19.(38) Considine, T.; Geary, S.; Kelly, A. L.; McSweeney, P. L. H.Proteolytic specificity of cathepsin G on bovine αs1- and β-caseins.Food Chem. 2002, 76, 59−67.(39) Considine, T.; Healy, A.; Kelly, A. L.; McSweeney, P. L. H.Hydrolysis of bovine caseins by cathepsin B, a cysteine proteinaseindigenous to milk. Int. Dairy J. 2004, 14, 117−124.(40) Moatsou, G.; Bakopanos, C.; Katharios, D.; Katsaros, G.;Kandarakis, I.; Taoukis, P.; Politis, I. Effect of high-pressure treatmentat various temperatures on indigenous proteolytic enzymes and wheyprotein denaturation in bovine milk. J. Dairy Res. 2008, 75, 262−269.(41) Larsen, L. B.; Hinz, K.; Jorgensen, A. L.; Moller, H. S.; Wellnitz,O.; Bruckmaier, R. M.; Kelly, A. L. Proteomic and peptidomic study ofproteolysis in quarter milk after infusion with lipoteichoic acid fromStaphylococcus aureus. J. Dairy Sci. 2010, 93, 5613−5626.(42) Gaucher, I.; Molle, D.; Gagnaire, V.; Gaucheron, F. Effects ofstorage temperature on physico-chemical characteristics of semi-skimmed UHT milk. Food Hydrocolloids 2008, 22, 130−143.(43) Farrell, H. M., Jr.; Brown, E. M.; Hoagland, P. D.; Malin, E. L.Higher Order Structures of the Caseins: A Paradox? In Advanced DairyChemistry1 Proteins; Fox, P. F., McSweeney, P. L. H., Eds.; Springer:New York, 2003; pp 203−231.(44) Berglund, L.; Andersen, M. D.; Petersen, T. E. Cloning andcharacterization of the bovine plasminogen cDNA. Int. Dairy J. 1995,5, 593−603.(45) Rollema, H. S.; Visser, S.; Poll, J. K. Spectrophotometric assay ofplasmin and plasminogen in bovine milk. Milchwissenschaft 1983, 38,214−217.(46) Marchin, S.; Putaux, J. L.; Pignon, F.; Leonil, J. Effects of theenvironmental factors on the casein micelle structure studied by cryotransmission electron microscopy and small-angle X-ray scattering/ultrasmall-angle X-ray scattering. J. Chem. Phys. 2007, 126, 045101.(47) Dalgleish, D. G. On the structural models of bovine caseinmicelles-review and possible improvements. Soft Matter 2011, 7,2265−2272.(48) Trejo, R.; Dokland, T.; Jurat-Fuentes, J.; Harte, F. Cryo-transmission electron tomography of native casein micelles frombovine milk. J. Dairy Sci. 2011, 94, 5770−5775.(49) Crudden, A.; Afoufa-Bastien, D.; Fox, P. F.; Brisson, G.; Kelly,A. L. Effect of hydrolysis of casein by plasmin on the heat stability ofmilk. Int. Dairy J. 2005, 15, 1017−1025.(50) Manji, B.; Kakuda, Y. The Role of Protein Denaturation, Extentof Proteolysis, and Storage Temperature on the Mechanism of AgeGelation in a Model System. J. Dairy Sci. 1988, 71, 1455−1463.(51) Kohlmann, K. L.; Nielsen, S. S.; Ladisch, M. R. Effects of a LowConcentration of Added Plasmin on Ultra-High TemperatureProcessed Milk. J. Dairy Sci. 1991, 74, 1151−1156.(52) Aroonkamonsri, J. The Role of Plasmin and BacterialProteinases on Age Gelation of UHT Milk (Pseudomonas f luorescens,Pseudomonas f ragi, Pseudomonas luteola, Vibrio f luvialis). Ph.D. Thesis,University of Guelph, Canada, 1996.(53) Newstead, D. F.; Paterson, G.; Anema, S. G.; Coker, C. J.;Wewala, A. R. Plasmin activity in direct-steam-injection UHT-processed reconstituted milk: Effects of preheat treatment. Int. DairyJ. 2006, 16, 573−579.(54) Matoba, T.; Hayashi, R.; Hata, T. Isolation of bitter peptidesfrom tryptic hydrolysate of casein and their chemical structure. Agric.Biol. Chem. 1970, 34, 1235−1243.(55) Hill, R.; Van Leeuwen, H. Bitter peptides from hydrolysedcasein coprecipitate. Aust. J. Dairy Technol. 1974, 29, 32−34.(56) Kim, H. O.; Li-Chan, E. C. Quantitative structure-activityrelationship study of bitter peptides. J. Agric. Food Chem. 2006, 54,10102−10111.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf502088u | J. Agric. Food Chem. 2014, 62, 6852−68606860
Supplementary data
1. Proteins in custom Mascot database used for identification of peptides from tandem MS
sp|P02662|CASA1A_BOVIN Alpha-S1-casein sp|P02662|CASA1B_BOVIN Alpha-S1-casein sp|P02662|CASA1C_BOVIN Alpha-S1-casein sp|P02662|CASA1D_BOVIN Alpha-S1-casein sp|P02662|CASA1G_BOVIN Alpha-S1-casein sp|P02662|CASA1I_BOVIN Alpha-S1-casein sp|P02663|CASA2A_BOVIN Alpha-S2-casein sp|P02663|CASA2B_BOVIN Alpha-S2-casein sp|P02663|CASA2D_BOVIN Alpha-S2-casein sp|P02666|CASBA1_BOVIN Beta-casein sp|P02666|CASBA2_BOVIN Beta-casein sp|P02666|CASBA3_BOVIN Beta-casein sp|P02666|CASBB_BOVIN Beta-casein sp|P02666|CASBC_BOVIN Beta-casein sp|P02666|CASBF_BOVIN Beta-casein sp|P02666|CASBI_BOVIN Beta-casein sp|P02668|CASKA_BOVIN Kappa-casein sp|P02668|CASKB_BOVIN Kappa-casein sp|P02668|CASKC_BOVIN Kappa-casein sp|P02668|CASKE_BOVIN Kappa-casein sp|P00711|LALBAA_BOVIN Alpha-lactalbumin sp|P00711|LALBAB_BOVIN Alpha-lactalbumin sp|P02754|LACBA_BOVIN Beta-lactoglobulin sp|P02754|LACBB_BOVIN Beta-lactoglobulin sp|P02754|LACBC_BOVIN Beta-lactoglobulin sp|P02754|LACBD_BOVIN Beta-lactoglobulin sp|P02769|ALBU_BOVIN Serum albumin sp|P02769|ALBU214_BOVIN Serum albumin sp|P24627|TRFL_BOVIN Lactotransferrin sp|P01239|PRL_BOVIN Prolactin sp|P80025|PERL_BOVIN Lactoperoxidase sp|P80025|PERLS_BOVIN Lactoperoxidase Ser394 variant sp|Q29443|TRFE_BOVIN Serotransferrin sp|P79345|NPC2_BOVIN Epididymal secretory protein E1 sp|Q9XSC9|TCO2_BOVIN Transcobalamin-2 sp|P18902|RET4_BOVIN Retinol-binding protein 4 sp|P15497|APOA1_BOVIN Apolipoprotein A-I sp|P18892|BT1A1_BOVIN Butyrophilin subfamily 1 member A1 sp|P80457|XDH_BOVIN Xanthine dehydrogenase/oxidase sp|P06868|PLMN_BOVIN Plasminogen sp|Q95114|MFGM_BOVIN Lactadherin sp|P31096|OSTP_BOVIN Osteopontin sp|Q8WML4|MUC1_BOVIN Mucin-1 sp|Q8MI01|MUC15_BOVIN Mucin-15 sp|Q8MI01-2|MUC15S_BOVIN Isoform 2 of Mucin-15 sp|Q9TUM6|PLIN2_BOVIN Perilipin-2 isoform-1 sp|Q9TUM6-2|PLIN2_BOVIN_2 Isoform 2 of Perilipin-2
2. Supplementary spectra of peptides used for relative quantification
αS1-CN f(1-22)
UV and EIC chromatogram MS spectrum at peak top Comments/Contamination
23,0 23,5 24,0 24,5 25,0 25,5 26,0
Absorb
ance 2
14 n
m
0
200
400
600
800
1000
Retention time (min)
23,0 23,5 24,0 24,5 25,0 25,5 26,0
Inte
nsity
(AU
)
-1e+5
0
1e+5
2e+5
3e+5
m/z
400 600 800 1000 1200 1400 1600 1800 2000
Inte
nsity
(AU
)
5e+4
1e+5
2e+5
2e+5
1309.2459
873.1665
655.1266
Overlay from αS1 f(106-124)
αS1-CN f(106-124)
UV and EIC chromatogram MS spectrum at peak top Comments/Contamination
23,0 23,5 24,0 24,5 25,0 25,5 26,0
Absorb
ance 2
14 n
m
0
200
400
600
800
1000
Retention time (min)
23,0 23,5 24,0 24,5 25,0 25,5 26,0
Inte
nsity
(AU
)
-1e+5
0
1e+5
2e+5
3e+5
m/z
400 600 800 1000 1200 1400 1600 1800 2000
Inte
nsity
(AU
)
0,0
2,0e+4
4,0e+4
6,0e+4
8,0e+4
1,0e+5
1,2e+5
1129.5582
753.3740
565.2839
Overlay from αS1 f(1-22)
αS2-CN f(1-21)
UV and EIC chromatogram MS spectrum at peak top Comments/Contamination
17 18 19 20 21
Absorb
ance 2
14 n
m
0
200
400
600
800
Retention time (min)
17 18 19 20 21
Inte
nsity
(AU
)
-5e+4
0
5e+4
1e+5
2e+5
2e+5
m/z
400 600 800 1000 1200 1400 1600 1800 2000
Inte
nsity
(AU
)
0
2e+4
4e+4
6e+4
8e+4
1e+5
1374.5114916.6757
687.7559
Overlay with 550.3334+1
αS2-CN f(1-24)
UV and EIC chromatogram MS spectrum at peak top Comments/Contamination
17 18 19 20 21
Absorb
ance 2
14 n
m
0
200
400
600
800
Retention time (min)
17 18 19 20 21
Inte
nsity
(AU
)
-5e+4
0
5e+4
1e+5
2e+5
2e+5
m/z
400 600 800 1000 1200 1400 1600 1800 2000
Inte
nsity
(AU
)
0
10000
20000
30000
40000
1567.0994
1045.0690
784.0544
Overlay with β-CN f(106-113) (207.2661+2)
αS2-CN f(115-150)
UV and EIC chromatogram MS spectrum at peak top Comments/Contamination
21,0 21,5 22,0 22,5 23,0 23,5 24,0
Absorb
ance 2
14 n
m
0
200
400
600
800
1000
21,0 21,5 22,0 22,5 23,0 23,5 24,0
Inte
nsity
(AU
)
-1e+5
0
1e+5
2e+5
3e+5
4e+5
400 600 800 1000 1200 1400 1600 1800 2000
Inte
nsity
(AU
)
0
10000
20000
30000
40000
50000
60000
1431.9881
1074.2425
859.7979
Overlay with 613.9731+6 (unknown component)
β-CN f(1-28)
UV and EIC chromatogram MS spectrum at peak top Comments/Contamination
23,0 23,5 24,0 24,5 25,0 25,5 26,0
Absorb
ance 2
14 n
m
0
500
1000
1500
23,0 23,5 24,0 24,5 25,0 25,5 26,0
Inte
nsity
(AU
)
-2e+5
0
2e+5
4e+5
6e+5
8e+5
400 600 800 1000 1200 1400 1600 1800 2000
Inte
nsity
(AU
)
0,0
2,0e+4
4,0e+4
6,0e+4
8,0e+4
1,0e+5
1,2e+5
1,4e+5
1,6e+5
1740.2566
1160.1748870.3845
696.7094
Overlay with 794.3810+3 (unknown component)
β-CN f(29-48)
UV and EIC chromatogram MS spectrum at peak top Comments/Contamination
Col 1 vs Col 2 Col 1 vs Col 2
17,0 17,5 18,0 18,5 19,0 19,5 20,0
Absorb
ance 2
14 n
m
0
200
400
600
17,0 17,5 18,0 18,5 19,0 19,5 20,0
Inte
nsity
(AU
)
-5e+4
0
5e+4
1e+5
2e+5
2e+5
400 600 800 1000 1200 1400 1600 1800 2000
Inte
nsity
(AU
)
0,0
5,0e+4
1,0e+5
1,5e+5
2,0e+5
2,5e+5
1281.0849
780.5020
Overlay with 802.4830+1 (unknown component)
1
Paper IV
Rauh, V. M., Johansen, L. B., Ipsen, R., Paulsson, M., Larsen, L. B., & Hammershøj, M. (2014)
Protein lactosylation in UHT milk during storage measured by liquid chromatography-mass
spectrometry and furosine
Manuscript intended for International Journal of Dairy Technology
3
Protein Lactosylation in UHT Milk during Storage measured by Liquid Chromatography-
Mass Spectrometry and Furosine
Valentin M. Rauh*a,b
, Lene B. Johansena, Mette Bakman
a, Richard Ipsen
c, Marie Paulsson
d, Lotte
B. Larsenb
and Marianne Hammershøjb
a Arla Foods Strategic Innovation Centre, Rørdrumvej 2, DK-8220 Brabrand, Denmark
b Aarhus University, Dept. of Food Science, Faculty of Science and Technology, Blichers Allé 20,
DK-8830 Tjele, Denmark
c University of Copenhagen, Department of Food Science, Rolighedsvej 30, DK-1958 Frederiksberg
C, Denmark
c Department of Food Technology, Engineering and Nutrition, Lund University, SE-221 00 Lund,
Sweden
*Corresponding author. Tel: +4587332783; FAX: +4587466688
E-mail address: [email protected]
4
ABSTRACT 1
The initial stage of the Maillard reaction, protein lactosylation, occurs during the heat treat-2
ment of milk and continues during subsequent storage. We compared the initial lactosylation 3
as well as the rate of lactosylation of milk proteins during storage in UHT milk subjected to 4
directly or indirectly heat treatment using liquid chromatography (LC) coupled with electro 5
spray injection mass spectrometry (ESI-MS). Furosine content was used as an overall marker 6
to allow for a quantitative correlation of lactosylation measured by LC-ESI-MS in the UHT 7
milks. Protein lactosylation increased during the storage period of 6 months at 20°C. Both the 8
initial extent and the rate of lactosylation positively correlated with the number of lysine resi-9
dues in the different proteins. An exponential or linear correlation with furosine concentration 10
could be established for major and minor lactosylated proteins, respectively. 11
5
1. Introduction 12
During heat treatment and following storage of UHT milk, the Maillard reaction is one of the 13
major quality deterioration factors. The formation of the Amadori product ε-lactulosyllysine 14
between lactose and the free ε-amino group of lysine results in a decreased nutritional value 15
of the milk as the lysine will no longer be bioavailable and extensive lactosylation can also 16
reduce the digestibility of proteins (Dalsgaard et al., 2007). In the past, the extent of lactosyl-17
ation has mainly been studied using indirect methods, such as the measurement of furosine, 18
which is a product of the acid hydrolysis of lactulosyllysine (Erbersdobler and Somoza, 19
2007). More recently, mass spectrometry (MS) has been shown to be a powerful tool for the 20
analysis of milk protein lactosylation in both wet and dry systems (Siciliano et al., 2013). Pro-21
tein lactosylation in wet milk has been studied both in the casein fraction (Scaloni et al., 2002; 22
Johnson et al., 2011), in the native whey fraction (Czerwenka et al., 2006; Meltretter et al., 23
2009; Losito et al., 2007) and in indigenous phospho-peptides (Pinto et al., 2012). Heat 24
treatment of milk has a major impact on the whey protein fraction and severe heat treatments, 25
such as e. g. UHT (136-140 °C for 2-6 s), result in an almost complete denaturation of the 26
whey proteins (Datta et al., 2002). In previous studies, the degree of lactosylation of native 27
whey proteins could be correlated with whey protein denaturation and fluorescence 28
(Meltretter et al., 2009; Losito et al., 2010). Despite the fact that caseins are the quantitatively 29
major milk proteins, lactosylation has only been observed for the most abundant caseins: αS1-30
casein (αS1-CN) and β-casein (β-CN). A quantitative correlation in wet milk systems with fu-31
rosine concentration could only be achieved for a singly lactosylated β-CN in processed 32
cheese (Steffan et al., 2006). In dry milk systems, a quantitative relationship between furosine 33
concentration and lactosylated peptides originating from whey proteins was established for 34
milk powder (Le et al., 2013). 35
Furthermore, the Amadori product is continuously formed during storage of milk. The devel-36
opment of lactulosyllysine during the storage is therefore an important parameter for shelf life 37
6
estimation of UHT milks. To our knowledge the development of protein lactosylation during 38
storage has not been studied using mass spectrometry before. 39
The aim of this study was to follow the lactosylation of the casein and whey fractions in di-40
rectly and indirectly heat treated UHT milks by ESI-MS and furosine analysis during subse-41
quent storage. In particular, the rate of lactosylation of different caseins and whey proteins 42
during storage was compared. 43
44
2. Materials and Methods 45
2.1 Materials 46
One day old low pasteurized milk (72 °C for 15 s) with 1.5 % fat was obtained from Arla 47
Foods Stockholm Dairy (Stockholm, Sweden) and processed in the UHT pilot plant (SPX, 48
Silkeborg, Denmark) at the Arla Strategic Innovation Center (Stockholm, Sweden). The milk 49
(200 L) was pre-heated at 95 °C for 5 s (Direct A) or 180 s (Direct B) in a tubular heat ex-50
changer before being subjected to the direct steam infusion heat treatment ( >150 °C for <0.2 51
s). After flash cooling, the milk was homogenized using a two-stage homogenizer (160/40 52
bar), further cooled down to 20 °C by a tubular heat exchanger and packed aseptically in 300 53
mL glass bottles. For the indirect UHT process (Indirect), milk was heated to 80 °C, homoge-54
nized using a two-stage homogenizer (160/40 bar) and further heated at 140 °C for 4 s. The 55
milk was cooled down to 20 °C by a tubular heat exchanger and packed aseptically in 300 mL 56
glass bottles. These trials were repeated three times during three consecutive weeks and the 57
milk was stored in the dark at 20 °C in a climate chamber for 6 months. One 300 mL bottle 58
per treatment was collected once every month for analysis. 59
60
2.1 Analytical methods 61
Protein content was measured by a MilkoScan FT 120 (Foss, Hillerød, Denmark). Analysis of 62
protein composition in the milk was performed as described by Bonfatti, Grigoletto, Cecchi-63
7
nato, Gallo, and Carnier (2008) with the following modifications. A sample of 200 µL of milk 64
(w/w) was frozen at -20 °C until further use. Disulphide bonds were reduced by the addition 65
of 20 µL 0.5 M dithiothreitol and 1 mL 100 mM tri-sodium citrate, 6 M urea at 30 °C for 60 66
min. The samples were centrifuged at 9300 x g at 5 °C for 10 min. From the clear phase, 200 67
µL were used for the analysis. A volume of 5 µL was injected into the LC-MS system. The 68
column was a BioSuiteTM
C18 PA-B (C18, 3.5 µm 2.1 mm x 250 mm, Waters, Milford, MA, 69
USA). Buffer A was Milli-Q water with 0.05 % trifluoroacetic acid (TFA) (v/v) and buffer B 70
acetonitrile with 0.1 % (v/v) TFA. Column temperature was 47 °C and a linear gradient from 71
34.8 % to 46.5 % buffer B from 2 to 16.5 min was applied at a flow rate of 0.35 mL min-1
72
with UV detection at 214 nm. MS detection was performed with a electrospray-time-of-flight 73
mass spectrometer with (ESI-TOF-MS; Agilent 6230 Aqurate mass detector, Agilent Tech-74
nologies) and MS scans were continuously recorded between a mass to charge ratio (m/z) of 75
300 and 3200 in 2 GHZ extended dynamic positive mode. The TOF-MS had a drying gas 76
temperature at 325 °C at a flow rate of10 L min-1
and a nebulizer pressure of 35 psi. Sheath 77
gas temperature was 300 °C at a flow rate of 7 L min-1
. Fragmentor energy was set 200 V and 78
the applied energy to the capillary was 3500 V. Data analysis was performed with Mass 79
Hunter software (Agilent Technologies, Santa Clara, CA, USA). 80
Mass spectra and UV chromatograms were analyzed by MassHunter (version B 6.01) and 81
Profinder (pre-release version B.06.00 Service Pack 1) software (Agilent Technologies, Santa 82
Clara, CA, USA). 83
Analysis of furosine was performed as follows: One mL milk sample was mixed with 3 mL of 84
10 M HCl in screw-cap tubes. Nitrogen was bubbled through the samples for 2 min to mix the 85
samples. The tubes were closed and heated at 115 ± 1 °C for 18 h. After heating, the samples 86
were cooled and filtered through a paper filter (pore size 20 µm). The filtrate was further fil-87
tered through a 0.45 µM disposable filter, diluted 5 times in 3 M HCl and transferred into 88
HPLC vials. For furosine analysis, 10 µL of the filtrate were injected into a Dionex 300DX 89
8
system (Dionex, Germering, Germany) with a DAD detector (UV at 280 nm) using a Supelco 90
Supelcosil LC-8 column for isocratic separation with 0.06 M sodium acetate as buffer at a 91
flow rate of 1 mL min-1
. 92
All measurements were performed in duplicate. Standard deviation between replicates for all 93
measurements was ranging from 5-10 %. 94
95
2.1 Data analysis 96
In order to achieve the optimal results, the ESI mass spectra were analyzed by 3 different 97
methods of data analysis for αS1-CN 8P. The αS1-CN 8P was chosen, as it is the most abun-98
dant casein and MS analysis of the milk showed that αS1-CN 8P was present in only one ge-99
netic in contrast to β-CN. Extracted ion chromatograms (EIC) of unmodified, mono-100
lactosylated and di-lactosylated αS1-CN were extracted using the most abundant ion as quanti-101
fier and the second abundant ion as qualifier (the used quantifier ions are shown in Figure 2). 102
The mass error was set to 100 ppm. Mass spectra were deconvoluted in MassHunter using the 103
maximum entropy setting (mass step 1 Da, signal to noise > 30 counts, minimum consecutive 104
charge states 5). Finally, the mass spectra were analyzed using the Profinder software, a batch 105
recursive deconvolution tool. A large molecular extractor algorithm (LMFE) was used. Reten-106
tion time tolerance was set to 1 min and an m/z window of 50 ppm. All EIC from proteins 107
having 5 to 35 charge states were smoothed using a Gaussian function before integration. 108
Additionally, the UV signal at 214 nm was integrated. Due to a co-elution of αS1-CN 8P and 109
9P, both variants were integrated as one peak. 110
Results are shown as mean value ± standard deviation of the 3 replicates. Linear regression 111
analysis and ANOVA was performed with Matlab software (Mathworks, Natick, MA). Mod-112
el? Class variables were heat treatment (1, 2, 3), storage time points (1- 6 months)? Differ-113
ences were regarded significant at 95%-level i.e. (P<0.05). 114
115
9
3. Results and Discussion 116
3.1 Initial furosine concentration and whey protein denaturation 117
In order to evaluate the effect of heat treatment, the initial furosine concentration and degree 118
of whey protein denaturation was analyzed in the different UHT milks after one day of stor-119
age (Table 1). As expected, the degree of whey protein denaturation increased with increasing 120
severity of the heat treatment. As previously reported, the degree of denaturation of β-lg was 121
higher compared to α-la (Dannenberg and Kessler, 1988). While there is a clear difference of 122
the heat treatment in respect to α-la denaturation for all milks, the degree of β-lg denaturation 123
in the Direct B and Indirect was not significantly different. This can be attributed to the al-124
most complete denaturation of β-lg in the milk (Table 1). As expected, the initial furosine 125
concentrations in Direct A and Direct B were approximately 10-15 times lower compared to 126
Indirect and within the range of previously reported results (Datta et al., 2002). No furosine 127
could be detected in the low pasteurized feed. The furosine content in Direct A was slightly 128
higher than in Direct B and the pre heat treatment of 95 °C for 180 s of Direct B did not affect 129
the furosine concentration. This trend continued during the entire storage period (Figure 1). 130
The furosine content increased linearly in all UHT milks during the 6 months storage period 131
and no significant difference in the rate of formation between the milks was observed. This is 132
in line with previous findings (Corzo et al., 1994; Nangpal and Reuter, 1990) and indicates 133
that the severity of the heat treatment affects the initial extent of the lactosylation but not the 134
rate of formation during storage under similar conditions. The linear increase of furosine dur-135
ing storage in all milks further shows that the Maillard reaction was in its initial stage during 136
the entire storage period and that degradation of lactulosyllysine to intermediate Maillard re-137
action products, such as 5-hydroxy-2-methylfurfural (HMF), most likely only occurred to a 138
limited extent (Le et al., 2011). 139
140
141
10
3.2 Comparison of MS analysis 142
Analysis of protein lactosylation using ESI-MS was evaluated for αS1-CN 8 P using EIC, de-143
convolution and Agilent Profinder. A representative mass spectrum of αS1-CN 8 P in Indirect 144
after 6 months of storage is shown in Figure 2. The most abundant ion for αS1-CN 8 P was at a 145
charge state +22 and the 2 ions with a m/z shift of 14.6 and 29.2 represented the mono and di-146
lactosylated αS1-CN 8 P species. No ion for a tri-lactosylated αS1-CN 8 P could be found in the 147
mass spectrum. An EIC of αS1-CN 8 P using the ions shown in the inset of Figure 2 are shown 148
in Figure 3 A. As previously reported, the addition of lactose renders the protein more hydro-149
philic, which results in a shift to lower retention times and a broadening of the peaks 150
(Czerwenka et al., 2006; Losito et al., 2007). The EIC of lactosylated and especially di-151
lactosylated αS1-CN 8 P are broader compared to the unlactosyalted αS1-CN 8 P, which can be 152
caused by the lactosylation of different lysine residues, which can result in a change in the 153
retention time (Czerwenka et al., 2006). Figure 3B shows the corresponding UV signal of αS1-154
CN 8 P and 9 P of Indirect, seen as a shoulder on the right side of the αS1-CN 8 P peak. The 155
comparison of the UV signal after 0 and 6 months of storage of Indirect shows a decrease in 156
peak height and a broadening of the peak due to the changes in retention time observed in the 157
EIC. The deconvoluted spectra of αS1-CN 8 P after 0 and 6 months of storage are shown in 158
Figure 4. After 6 months of storage, a tri-lactosylated αS1-CN 8 P could be observed in the 159
UHT milk and the intensity distribution shifted towards higher degrees of lactosylation (Fig-160
ure 4B). The decrease in un-lactosylated αS1-CN 8 P intensity was not proportional to the in-161
crease in the intensity of the lactosylated αS1-CN 8 P species. The UV area of αS1-CN 8 P on 162
the other hand was constant during the entire storage period in all UHT milks (± 6 %). This 163
relationship was valid for all l reduced proteins and the summed MS area decreased by ap-164
proximately 25-40 % after 6 months of storage. The decrease in intensity indicates a reduced 165
ionizability for lactosylated proteins, which previously are reported for caseins by Scaloni et 166
al. (2002) using a single quadrupole MS (Scaloni et al., 2002). For pH 4.6 soluble un-167
11
lacostylated and lactosylated β-lg and α-la on the other hand, an identical ionizability is sug-168
gested for matrix assisted laser desorption (MALDI) TOF-MS and triple quadrupole MS 169
(Czerwenka et al., 2006; Meltretter et al., 2009). A shift in the charge state distribution to-170
wards lower charge states occurred upon lactosylation of pH 4.6 soluble β-lg analysed with 171
triple quadrupole MS, which might be caused by a hindrance of protonation by the added lac-172
tose to lysine residues (Czerwenka et al., 2006). The reported differences in the ionization of 173
un-lactosylated and lactosylated milk proteins could be ascribed to the different MS instru-174
mentation and analysis conditions used. It is likely that the ionizability of lactosylated pro-175
teins could depend on the charge state distribution of the protein. A reduced ionization of lac-176
tosylated proteins might be negligible for lower charge state distribution or singly charged 177
proteins in MALDI-TOF-MS as there are other ionizable residues present in excess. However, 178
the lactosylation might have a substantial impact on the ionization of reduced and unfolded 179
proteins with a higher charge state distribution as observed in this study. A direct comparison 180
of the lactosylation of denatured and pH 4.6 soluble whey proteins was not possible in this 181
study due to the high degree of whey protein denaturation in the UHT milks (Table 1). 182
Because of the decrease and variation in MS area (measured as counts per second), the area 183
distribution of the summed areas of un-lactosylated and lactosylated proteins was used for 184
calculating the extent of lactosylation. Analysis using deconvolution and the Profinder soft-185
ware gave similar results, while analysis using EIC showed higher degrees of lactosylation 186
due to a missing exclusion of background noise (see supplementary section). In comparison to 187
the deconvolution algorithm, Profinder resulted in an improved extraction of the ion enve-188
lopes due to the recursive workflow and was thus used for further analysis. 189
190
3.2 Kinetics of protein lactosylation during storage 191
Di-lactosylated (for α-la and κ-CN) and tri-lactosylated proteins (for β-lg, αS1-CN, αS2-CN 192
and β-CN) were not present in the one day old UHT milks and accounted for < 2 % of the ar-193
12
ea in the most severely lactosylated UHT milk samples, i.e. Indirect after 6 months of storage, 194
and were not considered in the data analysis. A comparison of the absolute and relative area 195
distribution of αS1-CN 8 P lactosylation during storage in the UHT milks is shown in Figure 5 196
(for other proteins see supplementary section). As discussed above, the overall area decreased 197
during storage. In Indirect, both the area of un-lactosylated and mono-lactosylated αS1-CN 8 P 198
decreased during storage. The relative change in intensity of un-lactosylated and lactosylated 199
αS1-CN 8 P, on the other hand, fitted well to a linear regression in all milks (Figure 5 bottom 200
row). On a closer look, both the relative area of un-lactosylated and mono-lactosylated αS1-201
CN 8 P seemed to level out due to the difference in ionization efficiency of the lactosylated 202
αS1-CN 8 P and could also be fitted to an exponential function. In order to compare the lacto-203
sylation rates of different proteins and to reduce the number of variables, a linear regression 204
was applied. 205
Table 2 shows the results of the linear regression of casein lactosylation during storage. For 206
αS1-CN 8 P, β-CN A2 and κ-CN, the degree of lactosylation was significantly higher in Direct 207
B compared to Direct A, showing that the measurement of lactosylation by LC-MS is a very 208
sensitive method for discrimination between heat treatments. As the initial furosine results 209
already indicated, the initial degree of protein lactosylation in Direct A and Direct B was 210
similar for most of the proteins. The degree of lactosylation measured by LC-MS after heat 211
treatment in Indirect was only 2-4 times higher than in Direct A and Direct B. The lower de-212
gree of lactosylation found, compared to the furosine measurement (10-15 time higher; Figure 213
1), could be caused by the different response factors of the lactosylated proteins and the fact 214
that the two lactose moieties in di-lactosylated proteins were not taken into account. The ini-215
tial extent of lactosylation for the different caseins was αS2-CN > αS1-CN > β-CN > κ-CN, 216
which matches the number of lysine residues (24, 14, 11 and 9) and thus the available reaction 217
sites of the caseins. The results do, to some extent, support previous findings by Scaloni et al. 218
suggesting αS1-CN and β-CN being the most lactosylated caseins (Scaloni et al., 2002). 219
13
Similar to the initial casein lactosylation, the rate of lactosylation was alike for Direct A and 220
Direct B with the exception of β-CN A2. The decrease in un-lactosylated and increase in 221
mono-lactosylated protein was more pronounced in the Direct A and Direct B than indirect. A 222
higher degree of initial lactosylation resulted in a lower rate of lactosylation. This can be as-223
cribed to the slightly exponential nature of the curves (see Figure 5). The increase in di-224
lactosylated proteins on the other hand was significantly higher in Indirect reflecting the more 225
advanced degree of lactosylation in this UHT milk. The αS1-CN 8 P showed the highest rate 226
of lactosylation in all UHT milks followed by β-CN, αS2-CN and κ-CN. The lower rate of lac-227
tosylation of αS2-CN was ascribed to a high initial degree of lactosylation and reduced re-228
sponse factors of the lactosylated αS2-CN. Additionally, the amount of phosphorylations of 229
αS2-CN varies, which results in a massive overlay of the differently phosphorylated and lacto-230
sylated αS2-CN variants. This limits the extraction of ion envelopes and may cause ion sup-231
pression. This was also reflected in the lower R2–value of the linear regression of αS2-CN. The 232
rate of lactosylation for κ-CN was not significantly different between the UHT milks. This 233
could be caused by the low initial degree of lactosylation and the absence of multi-234
lactosylated variants with accordingly lower response factors. To our knowledge, the lacto-235
sylation of αS2-CN and κ-CN has not been reported previously. 236
The lactosylation of whey proteins during storage is shown in Table 3. The initial lactosyla-237
tion and rate of lactosylation of the reduced whey proteins was similar to the lactosylation of 238
the reduced caseins. The initial lactosylation as well as the rate of lactosylation during storage 239
of β-lg were higher compared to those of α-la, which correlate with the amount of available 240
reaction sites on the proteins with 15 lysine residues in β-lg and 12 in α-la, and is in line with 241
previous findings (Czerwenka et al., 2006; Losito et al., 2007). The initial degree of lactosyla-242
tion of α-la was significantly different for all UHT milks, but as with κ-CN, there was no sig-243
nificant difference in the rate of lactosylation of α-la between the UHT milks. Previous stud-244
ies indicate a difference in the extent of lactosylation of native and unfolded β-lg. The lysine 245
14
residues in unfolded β-lg could be easier accessible to react with lactose, and di-lactosylated 246
β-lg is the predominant form in the unfolded β-lg population (Losito et al., 2010). The rate of 247
lactosylation for β-lg during storage showed a similar trend, with a positive correlation of 248
rates of formation of dilatosylated β-lg and severity of the heat treatment and whey protein 249
denaturation (Table 3). However, the same trend was found for the caseins as well with the 250
exception of κ-casein. The caseins do not possess any notable tertiary structure, which would 251
explain this phenomenon. Thus, it cannot be concluded, whether the higher degree of whey 252
protein denaturation or simply a higher initial degree of lactosylation caused the higher rate of 253
formation of di-lactosylated β-lg in this study. 254
255
3.3 Correlation of lactosylation measured by furosine and LC-MS 256
The correlation of furosine concentration and decrease in un-lactosylated reduced protein is 257
shown in Figure 6 for β-CN, αS1-CN 8 P, α-la and β-lg. For less lactosylated proteins, such as 258
β-CN, κ-CN and α-la linear correlations between decrease in un-lactosylated proteins and in-259
crease in furosine concentrations were found with correlation coefficients of R2=0.95, 0.92, 260
and 0.87, respectively. This is in line with Steffan et al. (2006), who found a linear correlation 261
between unmodified β-CN and furosine in processed cheese, although only a singly lactosyl-262
ated β-CN was detected (Steffan et al., 2006). For the higher lactosylated proteins αS1-CN 8 P 263
and β-lg, the best correlations were achieved with an exponential fit resulting in R2=0.93 and 264
R2=0.95, respectively. The difference was caused by the change in response factor and the 265
higher concentration of di-lactosylated variants. Figure 6 shows that the area distribution of 266
lactosylated milk proteins measured by LC-MS can be used to quantify the extent of protein 267
lactosylation in milk during storage. Meltretter et al. (2009) previously showed that the lacto-268
sylation of pH 4.6 soluble whey proteins correlated with several heat markers, but the limiting 269
factor for this method is the amount of pH 4.6 soluble whey proteins, which does not allow 270
assessment of severely heat treated products (Meltretter et al., 2009). Furthermore, a potential 271
15
higher degree of lactosylation for denatured whey proteins compared to native whey proteins 272
might lead to an underestimation of the degree of lactosylation when measuring on pH 4.6 273
soluble whey proteins. The analysis of reduced caseins and whey proteins in this study, on the 274
other hand, did not have this limitation. For highly heat treated products with higher degrees 275
of lactosylation compared to the UHT milk used in this study, the less lactosylated proteins 276
(β-CN, κ-CN and α-la) could serve as marker proteins, since the effect of reduced intensity of 277
lactosylated species would be less pronounced. An improved chromatographic separation 278
could further facilitate the extraction of the MS spectra due to a reduced overlap of the differ-279
ent lactosylated species. The use of a relative area distribution of un-lactosylated and lactosyl-280
ated protein species additionally makes this method robust against other protein modifica-281
tions, such as oxidation, and limited proteolysis. It should be noted that the described method 282
is only suited for analysis of the initial stage of the Maillard reaction. For assessment of prod-283
ucts containing intermediate and advanced Maillard reaction products, the protein lactosyla-284
tion is not proportional to the extent of the Maillard reaction due to a degradation of lactulo-285
syllysine and different methods, such as measurement of advanced glycosylation end products 286
or fluorescence accumulation, should be applied (Le et al., 2013; Van Boekel, 1998; Bosch et 287
al., 2007; Le et al., 2011). 288
289
4. Conclusion 290
In conclusion, we could show that LC-MS is a suitable analysis to investigate the initial stage 291
of the Maillard reaction in UHT milk during storage. The results obtained by LC-MS allowed 292
a quantitative correlation with the commonly used marker, furosine. In comparison to the fu-293
rosine method, the LC-MS method allowed the analysis of the protein lactosylation of all ma-294
jor milk proteins, giving a more detailed view of the Maillard reaction in UHT milk during 295
storage. The extent of protein lactosylation in the directly heated UHT milk was approximate-296
ly 10-15 times lower compared to indirectly heated UHT milk. The rate of protein lactosyla-297
16
tion during storage was constant for indirect and directly heated UHT milk and correlated 298
with the number of lysine residues in the caseins and whey proteins. 299
300
5. Acknowledgements 301
The Danish Agency for Science, Technology and Innovation (DASTI) is thanked for financial 302
support of the study. We thank laboratory technicians Vibeke Mortensen and Betina Hansen 303
at the Arla Strategic Innovation Center for the assistance with the analysis and helpful discus-304
sions. Steve Madden and Steve Fischer at Agilent Technologies are thanked for expanding the 305
Profinder software, which enabled the analysis of intact proteins in this study. 306
307
6. References 308
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Datta, N, Elliott, A J, Perkins, M L and Deeth, H C (2002) Ultra-high-temperature (UHT) 322
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Nangpal, A and Reuter, H (1990) Reference diagram for furosine content in UHT milk. Kieler 348
Milchw. Forsch. 45 77-86. 349
Pinto, G, Caira, S, Cuollo, M, et al. (2012) Lactosylated casein phosphopeptides as specific 350
indicators of heated milks. Analytical and Bioanalytical Chemistry 402 1961-1972. 351
Scaloni, A, Perillo, V, Franco, P, et al. (2002) Characterization of heat-induced lactosylation 352
products in caseins by immunoenzymatic and mass spectrometric methodologies. Biochimica 353
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19
Tables
Table 1: Degree of whey protein denaturation and furosine content in UHT milks one day af-
ter processing. The values for β-lg are an average of β-lg A and B. Different superscripts in
one column indicate significant difference (P<0.05).
Milk Heat treatment Whey protein
denaturation [%]
Furosine
(mg/100 g protein)
β-lg α-la
Direct A 95°C/5 s +
> 150°C /< 0.2 s 86.6 ± 2.1
a 26.6 ± 4.0
a 11.6 ± 5.6
a
Direct B 95°C/180 s +
> 150°C /< 0.2 s 94.1 ± 1.6
b 45.5 ± 3.3
b 7.6 ± 4.6
a
Indirect 140 °C/4 s 93.6 ± 1.5 b 70.0 ± 6.1
c 119.9 ± 23.0
b
20
Table 2: Initial lactosylation and rate of lactosylation of reduced caseins in directly heated UHT milk (> 150 °C for < 0.2 s) pre-heated at 95 °C
for 5 s (Direct A) or 180 s (Direct B) and in indirect UHT milk (140°C for 4 s, Indirect) during storage at 20 °C. Results from linear regression
(y=ax+b) shown as average ± 95 % confidence interval. Different superscripts within a row for ‘slope a’ or within a row for ‘initial lactosylation
b’ indicate significant difference (P<0.05).
Protein Slope a (% month-1
) Initial lactosylation b (%) R2
Direct A Direct B Indirect Direct A Direct B Indirect Direct A Direct B Indirect
αS1-CN -4.210±0.209a -3.837±0.216
a -2.730±0.491
b 90.10±0.74
a 87.87±0.76
b 63.82±1.72
c 0.99 0.99 0.87
αS1-CN+lac 3.449±0.216a 3.110±0.221
a 1.631±0.380
b 8.19±0.76
a 9.59±0.78
a 28.67±1.12
b 0.98 0.98 0.85
αS1-CN+2lac 0.7609±0.055a 0.726±0.039
a 1.099±0.181
b 1.71±0.20
a 2.54±0.14
b 7.51±0.64
c 0.98 0.99 0.90
αS2-CN -2.293±0.299a -2.368±0.846
a -1.385±1.002
b 78.34±1.05
a 78.40±2.98
a 58.68±3.62
c 0.94 0.66 0.31
αS2-CN+lac 1.715±0.210a 1.794±0.582
ab 0.590±0.680
b 15.97±0.74
a 16.10±2.05
a 29.36±2.72
b 0.94 0.70 0.15
αS2-CN+2lac 0.578±0.136a 0.575±0.295
a 0.795±0.344
a 5.37±0.33
a 5.50±1.04
a 11.68±1.24
c 0.93 0.48 0.55
β-CN A1 -2.607±0.132
a -2.892±0.406
a -2.002±0.529
a 90.93±0.47
a 88.40±1.43
b 70.69±1.91
c 0.99 0.97 0.77
β-CN A1+lac 2.337±0.125
a 2.537±0.334
a 1.367±0.413
b 7.55±0.44
a 9.09±1.18
b 23.94±1.41
c 0.99 0.97 0.72
β-CN A1+2lac 0.270±0.036
a 0.355±0.082
a 0.635±0.135
b 1.52±0.13
a 2.51±0.29
b 5.37±0.49
c 0.93 0.92 0.84
β-CN A2 -2.534±0.162
ac -2.076±0.176
bc -1.944±0.614
abc 91.20±0.57
a 90.78±0.62
a 70.66±1.81
c 0.98 0.93 0.78
β-CN A2+lac 2.194±0.142
a 1.828±0.159
b 1.284±0.477
b 6.65±0.50
a 7.52±0.56
a 23.71±1.39
c 0.98 0.93 0.71
β-CN A2+2lac 0.340±0.045
a 0.247±0.036
b 0.660±0.146
c 2.16±0.16
a 1.69±0.13
b 5.72±0.51
c 0.93 0.82 0.83
κ-CN -1.543±0.112a -1.613±0.143
a -1.280±0.321
a 96.37±0.40
a 95.00±0.50
b 82.61±1.16
c 0.98 0.97 0.98
κ-CN+lac 1.543±0.112a 1.613±0.143
a 1.280±0.321
a 3.63±0.40
a 5.00±0.50
b 17.39±1.16
c 0.98 0.97 0.98
21
Table 3: Initial lactosylation and rate of lactosylation of whey proteins in directly heated UHT milk (> 150 °C for < 0.2 s) pre-heated at 95 °C for
5 (Direct A) or 180 s (Direct B) and in indirect UHT milk (140°C for 4 s, Indirect) during storage at 20 °C. Results from linear regression
(y=ax+b). Different superscripts within a row for ‘slope a’ or within a row for ‘initial lactosylation b’ indicate significant difference (P<0.05).
Protein Slope a (% month-1
) Initial lactosylation b (%) R2
Direct A Direct B Indirect Direct A Direct B Indirect Direct A Direct B Indirect
α-la -2.626±0.580a -2.352±0.199
a -1.686±0.491
a 95.74±1.99
a 91.80±0.70
b 71.85±1.77
c 0.85 0.97 0.73
α-la +lac 2.626±0.580a 2.352±0.199
a 1.686±0.491
a 4.26±1.99
a 8.20±0.70
b 28.15±1.77
c 0.85 0.97 0.73
β-lg A -4.032±0.461a -3.313±0.188
b -2.533±0.477
c 89.27±1.62
a 88.64±0.70
a 65.51±1.72
c 0.95 0.99 0.87
β-lg A+lac 2.893±0.462a 2.470±0.185
a 1.256±0.287
c 10.09±1.23
a 9.83±0.60
a 27.29±1.04
c 0.95 0.98 0.81
β-lg A+2lac 1.140±0.081a 0.842±0.081
b 1.277±0.207
c 1.14±0.28
a 1.53±0.30
a 7.20±0.73
c 0.98 0.96 0.90
β-lg B -4.004±0.257a -3.257±0.328
b -2.408±0.447
c 89.59±0.91
a 87.61±1.20
a 67.01±1.61
c 0.98 0.96 0.87
β-lg B+lac 3.355±0.281a 2.745±0.303
b 1.479±0.310
c 9.65±0.99
a 12.60±1.10
b 27.63±1.12
c 0.97 0.95 0.84
β-lg B+2lac 0.650±0.047a 0.512±0.064
b 0.929±0.148
a 0.76±0.17
a 1.08±0.20
a 5.36±0.53
c 0.98 0.94 0.90
Figures
Figure 1: Furosine development in directly heated UHT milk (> 150 °C for < 0.2 s) pre-heated at
95 °C for 5 (○) or 180 s (●) and in indirect UHT milk (140 °C for 4 s, □) during storage at 20 °C.
Figure 2: Summed mass spectra (retention time 11.0-13.2 min) of unmodified and lactosylated
αS1-CN 8 P in indirect UHT milk (140 °C for 4 s) after 6 months of storage at 20 °C. Inset is
showing a zoom of the most abundant ions. The m/z shifts due addition of lactose are indicated.
Figure 3: Extracted ion chromatogram of unmodified (solid line), mono-lactosylated (dashed line)
and di-lactosylated (dash-dot line) αS1-CN 8 P in indirect UHT milk (140 °C for 4 s) after 6
months of storage at 20 °C using the ions shown in Figure 2 (A). Corresponding UV signal at 214
nm of αS1-CN 8 P and 9 P after 0 (solid line) and 6 (dashed line) months of storage at 20 °C (B).
Figure 4: Deconvoluted mass spectrum of αS1-CN 8 P in indirect UHT milk (140 °C for 4 s) after
0 (A) and 6 (B) months of storage at 20 °C. Mass shifts due to addition of lactose are indicated.
Fig 5: Absolute areas (top row) and area distribution (bottom row) of unmodified and lactosylated
αS1-CN 8 P in directly heated UHT milk (> 150 °C for < 0.2 s) pre-heated at 95 °C for 5 (A) or
180 s (B) and in indirect UHT milk (140 °C for 4 s, C) during storage at 20 °C.
Figure 6: Correlation of furosine content and % of unmodified protein for α-la (A), β-lg A (B), β-
CN A2(C) and αS1-CN 8 P (D) from directly heated UHT milk (> 150 °C for < 0.2 s) pre-heated
at 95 °C for 5 (●) or 180 s (○) and in indirect UHT milk (140 °C for 4 s, □). The results of a line-
ar regression are shown.
Supplementary data 1. Summed mass spectra of unmodified and lactosylated caseins in indirect UHT milk (140 °C
for 4 s) after 6 months of storage at 20 °C. Inset is showing a zoom of the most abundant ions.
The m/z shifts due addition of lactose are indicated.
αS2-CN β-CN
κ-CN α-la
β-lg
2. Absolute and relative change in MS area of reduced αS2-CN during storage in Direct A (A),
Direct B (B) and Indirect (C).
3. Absolute and relative change in MS area of reduced β-CN during storage in Direct A (A), Di-
rect B (B) and Indirect (C).
3. Absolute and relative change in MS area of reduced κ-CN during storage in Direct A (A), Di-
rect B (B) and Indirect (C).
4. Absolute and relative change in MS area of reduced α-la during storage in Direct A (A), Direct
B (B) and Indirect (C).
5. Absolute and relative change in MS area of reduced β-lg during storage in Direct A (A), Direct
B (B) and Indirect (C).
6. Development of summed intensity of unmodified and lactosylated reduced αS1-CN 8 P using
EIC area, deconvolution intensity, profinder area and UV area at 214 nm of αS1-CN 8 P and 9 P
in indirect UHT milk (140 °C for 4 s) during storage at 20 °C.